Methods, compositions, and devices for information storage

ABSTRACT

The disclosure provides a novel system of storing information using a charged polymer, e.g., DNA, the monomers of which correspond to a machine-readable code, e.g., a binary code, and which can be synthesized and/or read using a novel nanochip device comprising nanopores; novel methods and devices for synthesizing oligonucleotides in a nanochip format; novel methods for synthesizing DNA in the 3′ to 5′ direction using topoisomerase; novel methods and devices for reading the sequence of a charged polymer, e.g., DNA, by measuring capacitive variance as the polymer passes through the nanopore; and further provides compounds, compositions, methods and devices useful therein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of International ApplicationNo. PCT/US2017/20044, filed Feb. 28, 2017, which claims priority to U.S.Provisional Patent Application No. 62/301,538, filed Feb. 29, 2016, andU.S. Provisional Patent Application No. 62/415,430, filed Oct. 31, 2016,and the contents of which are hereby incorporated by reference in theirentirety.

FIELD

The invention relates to novel methods, compositions and devices usefulfor information storage and retrieval, using nanopore devices tosynthesize and sequence polymers, e.g., nucleic acids.

BACKGROUND

There is a continuing demand to store ever more data on or in physicalmedia, with storage devices getting ever smaller as their capacity getsbigger. The amount of data stored is reportedly doubling in size everytwo years, and according to one study, by 2020 the amount of data wecreate and copy annually will reach 44 zetabytes, or 44 trilliongigabytes. Moreover, existing data storage media such as hard drives,optical media, and magnetic tapes, are relatively unstable and becomecorrupted after prolonged storage.

There is an urgent need for alternative approaches to storing largevolumes of data for extended periods, e.g. decades or centuries.

Some have proposed using DNA to store data. DNA is extremely stable andcould in theory encode vast amounts of data and store the data for verylong periods. See, for example, Bancroft, C., et al., Long-Term Storageof Information in DNA, Science (2001) 293: 1763-1765. Additionally, DNAas a storage medium is not susceptible to the security risks oftraditional digital storage media. But there has been no practicalapproach to implementing this idea.

WO 2014/014991, for example, describes a method of storing data on DNAoligonucleotides, wherein information is encoded in binary format, onebit per nucleotide, with a 96 bit (96 nucleotide) data block, a 19nucleotide address sequence, and flanking sequences for amplificationand sequencing. The code is then read by amplifying the sequences usingPCR and sequencing using a high speed sequencer like the Illumina HiSeqmachine. The data block sequences are then arranged in the correct orderusing the address tags, the address and flanking sequences are filteredout, and the sequence data is translated into binary code. Such anapproach has significant limitations. For example, the 96 bit data blockcould encode only 12 letters (using the conventional one byte or 8 bitsper letter or space). The ratio of useful information stored relative to“housekeeping” information is low—approximately 40% of the sequenceinformation is taken up with the address and the flanking DNA. Thespecification describes encoding a book using 54,898 oligonucleotides.The ink-jet printed, high-fidelity DNA microchips used to synthesize theoligonucleotides limited the size of the oligos (159-mers described wereat the upper limit). Furthermore, reading the oligonucleotides requiresamplification and isolation, which introduces additional potential forerror. See also, WO 2004/088585A2; WO 03/025123 A2; C. BANCROFT:“Long-Term Storage of Information in DNA”, Science (2001) 293 (5536):1763c-1765; COX J P L: “Long-term data storage in DNA”, Trends inBiotechnology (2001)19(7): 247-250.

DNA sequencing devices include nanopore-based devices from OxfordNanopore, Genia and others. In many of those devices, typically ananopore is used in a fluid-filled cell to read the DNA data bymeasuring a change in current as the DNA passes through the nanopore,which are typically in the range of nano-amps. Measurements based onchanges in capacitance have been proposed but are not commercial; thechanges are in the range of pico/fempto/atto-farads. Accordingly, it isvery difficult to reliably and repeatably detect such small changes, asthey are difficult to distinguish over typical background noise. Thedifficulties are further enhanced in that DNA can move through ananopore at the rate of approximately one million bases per second,which is too fast to read accurately using existing means, requiring theuse of protein nanopores which slow the passage of DNA through thenanopore, and which are impractical for reading large amounts of data.

Existing nano-pore based DNA data readers do not overcome these problemsand thus do not provide highly precise, repeatable, reliable, automated,and robust DNA data reading results. Thus, it would be desirable to havea device that provides high quality, reliable DNA data reading resultsand also provides a scalable approach to reliably read data stored onmultiple DNA molecules simultaneously.

While the potential information density and stability of DNA make it anattractive vehicle for data storage, as has been recognized for overtwenty-five years, there is still no practical approach to writing andreading large amounts of data in this form.

BRIEF SUMMARY

We have developed a new approach to nucleic acid storage, usingnanofluidic systems to synthesize the nucleic acid sequences andnanopore readers to read the sequences. Our approach allows for thesynthesis, storage and reading of DNA strands which are hundreds,thousands or even millions of bases long. Because the sequences arelong, only a relatively small proportion of the sequence is taken upwith identifying information, so that the information density is muchhigher than in the approach described above. Moreover, in someembodiments, the nucleic acid as synthesized will have a specificlocation on a nanochip, so the sequence can be identified even withoutidentifying information. The sequencing carried out in nanochambers isvery rapid, and reading the sequence through a nanopore can be extremelyrapid, on the order of up to one million bases per second. Since onlytwo base types are required, the sequencing can be faster and moreaccurate than sequencing procedures that must distinguish among fournucleotide base types (adenine, thymine, cytosine, guanine). Inparticular embodiments, the two bases will not pair with one another andform secondary structures and will also be of different sizes. Forexample, adenine and cytosine would be better for this purpose thanadenine and thymine, which tend to hybridize, or adenine and guanine,which are of similar size.

In some embodiments, this system can be used to synthesize long polymersencoding data, which can be amplified and/or released, and thensequenced on a different sequencer. In other embodiments, the system canbe used to provide custom DNA sequences. In still other embodiments, thesystem can be used to read DNA sequences.

The nanochips used in one embodiment contain at least two separatereaction compartments connected by at least one nanopore, which preventsat least some of the components from mixing, but allows as few as asingle molecule of DNA, or other charged polymers, e.g., RNA or peptidenucleic acid (PNA), to cross from one reaction compartment into anotherin a controllable manner. The transfer of the polymer (or at least theend of the polymer to which monomers are added) from one compartment toanother permits sequential manipulations/reactions to the polymer, suchas addition of bases, using enzymes which are prevented from crossingthrough the nanopore, for example because they are too large or becausethey are tethered to a substrate or bulky portion. Nanopore sensorsreport back on the movement or location of the polymer and its state,for example its sequence and whether the attempted reaction wassuccessful. This allows data to be written, stored, and read, forexample wherein the base sequence corresponds to a machine readablecode, for example a binary code, with each base or group of basescorresponding to a 1 or 0.

Accordingly, the invention includes, inter alia, the followingembodiments,

-   -   A nanochip for synthesis of an electrically charged polymer,        e.g., DNA, comprising at least two distinct monomers, the        nanochip comprising two or more reaction chambers separated by        one or more nanopores, wherein each reaction chamber comprises        an electrolytic fluid, one or more electrodes to draw the        electrically charged polymer into the chamber and one or more        reagents to facilitate addition of monomers or oligomers to the        polymer. The nanochip may optionally be configured with        functional elements to guide, channel and/or control the DNA, it        may optionally be coated or made with materials selected to        allow smooth flow of DNA or to attach the DNA, and it may        comprise nanocircuit elements to provide and control electrodes        proximate to the nanopores. For example, the one or more        nanopores may optionally each be associated with electrodes        which can control the movement of the polymer though the        nanopore and/or detect changes in electric potential, current,        resistance or capacitance at the interface of the nanopore and        the polymer, thereby detecting the sequence of the polymer as it        passes through the one or more nanopores. In particular        embodiments, the oligomers are synthesized using polymerases or        site specific recombinases. In some embodiments, the polymer is        sequenced during the course of synthesis, to allow for the        detection and optionally correction of mistakes. In some        embodiments, the polymer thus obtained is stored on the nanochip        and can be sequenced when it is desired to access the        information encoded in the polymer sequence.    -   Methods and devices for determining the sequence of a polymer,        e.g., DNA, in a nanopore chip by measuring the capacitive        variance in a resonant RF circuit as the DNA is drawn through        the nanopore by a DC bias.    -   A method of synthesizing a polymer, e.g., DNA, using a nanochip        as described.    -   A single stranded DNA molecule wherein the sequence consists        essentially of only nonhybridizing nucleotides, for example        adenine and cytosine nucleotides (As and Cs), which are arranged        in sequence to correspond to a binary code, e.g., for use in a        method of data storage.    -   A double stranded DNA comprising a series of nucleotide        sequences corresponding to a binary code, wherein the double        stranded DNA further comprises    -   A method of reading binary code encoded in DNA, comprising using        a nanopore sequencer.    -   A method of data storage and devices therefor, using the above        nanochip to make an electrically charged polymer, e.g., DNA,        comprising at least two distinct monomers, wherein the monomers        are arranged in sequence to correspond to a binary code.    -   Methods and systems for storing and reading data on a memory        string (such as DNA or a polymer) in situ in a nanopore-based        chip, includes providing a cell having at least three chambers,        having an Add “1” chamber arranged to add a “1” bit to the        polymer and an Add “0” chamber arranged to add a “0” bit to the        polymer, and a “deblock” chamber arranged to enable the polymer        to receive the “1” bit and “0” bit when the polymer enters the        Add “1” or Add “0” chambers, respectively, successively steering        the polymer from the “deblock” chamber through the nanopore to        the Add “1” chamber or to the Add “0” chamber based on a        predetermined digital data pattern to create the digital data        pattern on the polymer, and reading the digital data stored on        the polymer as it passes through the nanopore using a resonance        frequency response of a nanopore-polymer resonator (NPR) on the        chip.    -   Methods and systems for reading data stored in a polymer include        providing a resonator having an inductor and a cell, the cell        having a nano-pore and a polymer that can traverse through the        nanopore, the resonator having an AC output voltage frequency        response at a probe frequency in response to an AC input voltage        at the probe frequency, providing the AC input voltage having at        least the probe frequency, and monitoring the AC output voltage        at least at the probe frequency, the AC output voltage at the        probe frequency being indicative of the data stored in the        polymer at the time of monitoring, wherein the polymer includes        at least two monomers having different properties causing        different resonant frequency responses.

Further aspects and areas of applicability of the present invention willbecome apparent from the detailed description provided hereinafter. Itshould be understood that the detailed description and specificexamples, while indicating preferred embodiments of the invention, areintended for purposes of illustration only and are not intended to limitthe scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description and the accompanying drawings, wherein:

FIG. 1 shows a diagram of a simple two-chamber nanochip design, with adividing membrane perforated by a nanopore, and electrodes on eitherside of the membrane.

FIGS. 2 and 3 show how the charged polymer, e.g. DNA, is drawn towardsthe anode.

FIGS. 4 and 5 show that the polymer can be moved back by reversing thepolarity of the electrodes.

FIG. 6 shows a two chamber nanochip design for DNA synthesis, in which apolymerase enzyme is located in one chamber, a de-blocking enzyme is inthe other chamber, and neither can pass through the nanopore.

FIG. 7 shows addition of an adenine nucleotide when a 3′-blocked dATP(A) flows through left chamber, and the current is set ‘forward’ tobring the DNA into the chamber.

FIG. 8 shows deprotection of the oligonucleotide so an additionalnucleotide can be added. For example, deprotection occurs after movingthe DNA into the chamber by setting the current to ‘reverse’.

FIG. 9 shows addition of a 3′-blocked dCTP (C). In certain embodiments,fluid flow is used to exchange the contents of this chamber, e.g., asdepicted, previously there was ‘A’ in this chamber.

FIG. 10 shows how multiple separate retaining chambers can be providedwhile the flow chamber becomes a single lane to provide reagents.

FIG. 11 shows an approach to keeping the DNA associated with itschamber, by attaching to the chamber (upper DNA fragment in figure) orby coupling to a bulky group that cannot get through the nanopore (lowerDNA fragment in figure). In this system, the end of the DNA can stillmove into the flow chamber and receive additional nucleotides, but theother end remains in the retaining chamber.

FIG. 12 shows a configuration where the DNA is attached to the wall ofthe chamber and controlled by multiple electrodes.

FIG. 13 shows how the DNA can be retained in the chamber when desired,simply by controlling the polarity of the electrodes.

FIG. 14 shows an array with free-flowing reagents through both sides,with the DNA bound to the surface of a chamber.

FIG. 15 shows an alternate design with the electrodes on the sidesadjacent to the dividing membrane, which allows for less expensivemanufacture.

FIG. 16 shows a three-compartment arrangement, where the DNA can bemoved from compartment to compartment by the electrodes. This systemdoes not require significant flow of reagents during synthesis.

FIG. 17 shows an example of how reagents could be configured in a threecompartment arrangement.

FIG. 18 depicts an oligonucleotide tethered adjacent to a nanopore,where the nanopore has electrode elements on either side of themembrane.

FIG. 19 depicts a series of DNA molecules attached along a membranecomprising nanopores and each under control of electrodes adjacent to ananopore, with a flow lane on either side of the membrane. For example,as depicted, the left flow lane provides a flow of bufferwash/3′-blocked dATP (A)/buffer wash/3′-blocked dCTP (C)/buffer wash,wherein the DNA molecules are brought into the flow chamber only whenthe desired nucleotide is present. The right lane provides deblockingagent(s) to deprotect the 3′ end of the nucleotide and allow foraddition of another nucleotide. In one embodiment, the deblockingagent(s) flow when the left lane is being washed with buffer. In anotherembodiments, the deprotecting agent(s) are too bulky to cross to theleft lane via the nanopores.

FIGS. 20-22 depict the schematically the proof of concept experimentswherein the bits used to encode the data are short oligomers attachedusing topoisomerase.

FIG. 23 depicts a format for a nanopore sequencer wherein the polymersequence is read using capacitive variance. In this capacitive readoutscheme, electrodes form the top and bottom plates of a capacitor,separated by a membrane comprising a nanopore. The capacitor is embeddedin a resonant circuit, wherein a pulsating direct current can draw thecharged polymer through the nanopore. The change in capacitance ismeasured as the polymer, e.g. DNA, passes through the nanopore, usinghigh frequency impedance spectroscopy. A major advantage of thisapproach, particularly with DNA, is that the measurement frequency canbe very high (effectively a measurement for every cycle, so a 100 MHzfrequency corresponds to 100 million measurements per second), and muchgreater than the rate of transfer of monomers through the nanopore (DNA,for example, unless somehow constrained, will pass through the nanoporein response to electrical current at a speed on the order of 1 millionnucleotides per second).

FIG. 24 depicts a dual addition chamber layout, suitable for adding twodifferent types of monomers or oligomers, e.g., for 2-bit or binaryencoding. The upper part of the figure shows a top view. The lower partshows a side view cross-section. The full device in this embodiment canbe assembled from up to 3 independently fabricated layers and joined bywafer bonding, or may be formed by etching a single substrate. The chipcomprises an electrical control layer (1), a fluidics layer (2) whichcontains the two addition chambers atop a reserve chamber, with thecharged polymer (e.g., DNA) anchored between nanopore entrances to thefirst and second addition chambers, and an electrical ground layer (3).

FIG. 25 depicts the operation of the dual addition chamber layout ofFIG. 24. It will be observed that at the base of each addition chamber,there is a nanopore (4). The nanopore is made, for example, by drillingwith FIB, TEM, wet or dry etching, or via dielectric breakdown. Themembrane (5) comprising the nanopores is, e.g., from 1 atomic layer to10's of nm thick. It is made from, e.g., SiN, BN, SiOx, Graphene,transition metal dichalcogenides e.g. WS₂ or MoS₂. Underneath thenanopore membrane (5) there is a reserve or deblocker chamber (6), whichcontains reagents for deprotection of the polymer following addition ofa monomer or oligomer in one of addition chambers (it will be recalledthat the monomers or oligomers are added in end-protected form, so thatonly a single monomer or oligomer is added at a time). The polymer (7)can be drawn into or out of the addition chambers by changing thepolarity of the electrodes in the electrical control layer (1).

FIG. 26 depicts a top view of similar layout to FIGS. 24 and 25, buthere there are four addition chambers which share a common reserve ordeblocker chamber and the polymer is tethered at a position (9) withaccess to each of the four chambers. The cross section of this layoutwould be as depicted in FIGS. 24 and 25, and the charged polymer can bemoved into each of the four addition chambers by operation of theelectrodes in the electrical control layer (1 in FIG. 24).

FIG. 27 depicts a top view of a nanopore chip having multiple sets ofdual addition chambers as depicted in FIGS. 24 and 25, allowing multiplepolymers to be synthesized in parallel. The monomers are (here dATP anddGTP nucleotides represented as A and G) are loaded into each chambervia serial flow paths. One or more common deblocker flow cells allowsfor the polymers to be deprotected after addition of a monomer oroligomer in one of the addition chambers. This also allow the polymersto be detached on demand (for example using a restriction enzyme in thecase of DNA, or a chemical detachment from the surface adjacent to thenanopore, and collected externally. In this particular embodiment, thedeblocker flow cells are perpendicular to the fluidics loading channelsused to fill the addition chambers.

FIG. 28 depicts further details of the wiring for the dual additionchamber layouts. The electrical control layer (1) includes wiring madefrom metal or polysilicon. The wiring density is increased by 3Dstacking, with electrical isolation provided by dielectric deposition(e.g., via PECVD, sputtering, ALD etc). The contact (11) to the topelectrode by in the addition chamber in one embodiment is made usingThrough Silicon Via (TSV) by Deep Reactive Ion Etch (DRIE) (cryo orBOSCH process). Individual voltage control (12) allows for each additionchamber to be addressed individually, allowing fine control of thesequence of multiple polymers in parallel. The right side of the figuredepicts a top view illustrating wiring to multiple addition cells. Theelectrical ground layer (3) may be common (as shown) or split to reducecross coupling between the cells.

FIG. 29 depicts an alternative configuration where the controlelectrodes (13) for the addition chambers may be deposited on the sideof the chamber in a wrap around fashion instead of at the top of thechamber.

FIG. 30 depicts a SDS-PAGE gel confirming that topoisomerase additionprotocol as described in Example 3 works, with bands corresponding tothe expected A5 and B5 products being clearly visible.

FIG. 31 depicts an agarose gel confirming that the PCR product ofExample 5 is the correct size. Lane 0 is a 25 base pair ladder; lane 1is product of experiment, line corresponding to expected molecularweight; lane 2 is negative control #1; lane 3 is negative control #2;lane 4 is negative control #4.

FIG. 32 depicts an agarose gel confirming that the restriction enzyme asdescribed in Example 5 produces the expected product. The ladder on theleft is a 100 base pair ladder. Lane 1 is undigested NAT1/NAT9c, Lane 2is digested NAT1/NAT9c. Lane 3 is undigested NAT1/NAT9cI, Lane 4 isdigested NAT1/NAT9cI.

FIG. 33 depicts Immobilization of DNA near nanopore. Panel (1) shows DNAwith an origami structure on one end in the left chamber (in the actualnanochip, there initially are many such origami structures in the leftchamber). Panel (2) illustrates the system with anode on the right,which drives the DNA to the nanopore. While the DNA strand is able totransit the nanopore, the origami structure is too large to passthrough, so the DNA is ‘stuck’. Turning the current off (panel 3) allowsthe DNA to diffuse. With suitable chemistry, the end of the DNA strandis able to bind when it comes in contact with the surface near thenanopore. In panel (4) a restriction enzyme is added, which cuts theorigami structure from the DNA. The chamber is washed to remove enzymeand residual DNA. The final result is a single DNA molecule attachednear a nanopore, able to be moved back and forth through the nanopore.

FIG. 34 depicts a basic functioning nanopore. In each panel, the y-axisis current (nA) and the x-axis is time (s). The left panel “Screening ofRF Noise” illustrates the utility of the Faraday cage. A chip with nonanopore is placed in the flow cell and 300 mV applied. When the lid ofthe Faraday cage is closed (first arrow) the noise reduction can beseen. A small spike occurs when the latch is closed (second arrow).Notice the current is ˜0 nA. After pore manufacture (middle panel),application of 300 mV (arrow) results in a current of ˜3.5 nA. When DNAis applied to the ground chamber and +300 mV is applied DNAtranslocations (right panel) can be observed as transient decreases inthe current. (Note, in this case the TS buffer is used: 50 mM Tris, pH8, 1M NaCl). Lambda DNA is used for this DNA translocation experiment.

FIG. 35 depicts a simplified picture illustrating the main features ofthe DNA origami structure: a large single stranded region, the cubicorigami structure, and the presence of 2 restriction sites (SwaI andAlwN1) near the origami structure.

FIG. 36 depicts an electron microscope image of the manufactured DNAorigami structure, and demonstrates the expected topology. Origami ismade in 5 mM Tris base, 1 mM EDTA, 5 mM NaCl, 5 mM MgCl2. In order tomaintain the origami structure, it is preferable to have Mg⁺⁺concentrations of ˜5 mM or Na⁺/K⁺ concentrations around 1M. The origamistructure is stored at 4° C. at 500 nM.

FIG. 37 depicts a restriction digestion of the DNA origami to confirmcorrect assembly and function. The lane on the far left provides MWstandards. The restriction sites are tested by digesting the origamiwith AlwN1 and Swa1. The four test lanes contain reagents as follows(units are microliters):

1 2 3 4 origami 10  10  10  10 SwaI — 1 — 1 AlwNI — — 1 1 NEB 3.1 10x 22 2 2 water 8 7 7 6

Test lane (1) is a negative control; (2) is digestion with Swa1; (3) isdigestion with AlwN1; (4) is double digestion with Swa1/AlwN1. Digestionis performed at room temperature for 60 minutes, followed by 37° C. for90 minutes. Agarose gel ½×TBE-Mg (½×TBE with 5 mM MgCl2), visualizedwith ethidium bromide staining. Individual digestion with either enzymeshows no mobility effect in a gel, but digestion with both enzymestogether (lane 4) results in two fragments of different lengths, asexpected.

FIG. 38 depicts binding of biotin-labeled oligonucleotides tostreptavidin-coated beads vs. binding to control BSA coated beads. They-axis is fluorescence units, ‘pre-binding’ is oligo fluorescence fromtest solution prior to binding beads, (−) controls are fluorescence seenafter binding to two different batches of BSA-conjugated beads, SA-1 andSA-2 are fluorescence seen after binding to 2 different batches ofstreptavidin-conjugated beads. A small apparent amount of binding isobserved with BSA-conjugated beads, but much larger binding is seen withthe streptavidin-conjugated beads.

FIG. 39 depicts binding of biotin-labeled oligonucleotides tostreptavidin-coated beads vs. binding to control BSA coated beads indifferent buffer systems, MPBS and HK buffer. The left bar ‘Neg Ctrl’ isthe oligo fluorescence from test solution prior to binding the beads.Middle column shows fluorescence of ‘BSA beads’ and right column of ‘SAbeads’ after binding to BSA or streptavidin beads respectively. In bothbuffer systems, the fluorescence is reduced by the streptavidin beadsrelative to controls, indicating that the biotin-labeledoligonucleotides are binding well to streptavidin-coated beads indifferent buffer systems.

FIG. 40 depicts a functioning conjugated SiO₂ nanopore, wherein thesurface is strepavidin coated on one side and BSA coated on the other.The x-axis is time and the y-axis is current. The dot shows the pointwhere the current is reversed. There is a brief overshoot when thecurrent is reversed, then the current settles to approximately the sameabsolute value. The nanopore shows a current of ˜+3 nA at 200 mV and ˜3nA at ˜200 mV.

FIG. 41 shows a representation of an origami DNA structure inserted intoa nanopore.

FIG. 42 shows a representation of attachment of the single stranded DNAto the streptavidin-coated surface adjacent to the nanopore.

FIG. 43 shows experimental results of an origami DNA attached to thesurface near a nanopore. Current is + or −˜2.5 nA in both directions,which is less than the original current of +/−˜3 nA, reflecting partialobstruction by the origami structure. The x-axis is time (s), y-axis iscurrent (nA), circles represent voltage switch points.

FIG. 44 shows the insertion of origami DNA, resulting in a slight dropin current. The origami immediately exits the nanopore when the currentis released. The x-axis is time (s), y-axis is current (nA), circlesrepresent voltage switch points.

FIG. 45 shows a representation of controlled movement of a DNA strandback and forth through a nanopore by application of current. On the leftside the DNA is in the pore, so the observed current will be lower thanif there was no DNA in the pore. When the current is reversed (rightside) the is no DNA in the pore so the current will be unchanged.

FIG. 46 shows experimental results confirming this representation. Whena positive voltage is applied the current is ˜3 nA, comparable to thecurrent typically observed when the pore is open. When the voltage isreversed the current is ˜−2.5 nA. This is lower than the currenttypically seen when the pore is open, and corresponds to the currenttypically observed when the pore is blocked by a strand of DNA. Severalsequential voltage switches show consistent results, suggesting that theDNA is alternating in configuration as depicted in FIG. 45.

FIG. 47 shows different conjugation chemistries to link the DNA to thesurface adjacent to the nanopore.

FIGS. 48A, 48B, and 48C are three views of a polymer and a nanopore andthe equivalent circuit, in accordance with embodiments of the presentinvention.

FIG. 49A is an equivalent circuit for a resonator made with a nanoporecell, in accordance with embodiments of the present invention.

FIG. 49B is a graph of magnitude and phase of the output response of theresonator of FIG. 49A, in accordance with embodiments of the presentinvention.

FIG. 50 is a family of curves showing a range of magnitude and phase ofthe output responses of the resonator of FIG. 49A, in accordance withembodiments of the present invention.

FIG. 51 is a time series showing a polymer passing through a nanoporeand the resulting resonator magnitude and phase of the output responsesat a probe frequency, in accordance with embodiments of the presentinvention.

FIG. 52 is a time series showing a polymer passing through a nanoporeand the resulting resonator magnitude and phase of the output responsesat a second probe frequency, in accordance with embodiments of thepresent invention.

FIG. 53 is an equivalent circuit of a plurality of parallelnanopore-polymer resonators and signal processing, in accordance withembodiments of the present invention.

FIG. 54 is a frequency plot for several resonant frequency bandwidths,in accordance with embodiments of the present invention.

FIG. 55A are frequency plots for AC input voltages Vin, in accordancewith embodiments of the present invention.

FIG. 55B are time & frequency plots for alternative AC input voltagesVin, in accordance with embodiments of the present invention.

FIG. 56 are magnitude and phase frequency plots at three probefrequencies, in accordance with embodiments of the present invention.

FIG. 57 is a block diagram of a 2D array of nanopore-polymer resonators,in accordance with embodiments of the present invention.

FIG. 58 is a side cross-sectional view of a nanopore memory chip, inaccordance with embodiments of the present invention.

FIG. 59 is a top view of an inductor used in the chip of FIG. 58, inaccordance with embodiments of the present invention.

FIG. 60 is an equivalent circuit diagram of a “bias-tee” configurationto connect both AC and DC signals, in accordance with embodiments of thepresent invention.

FIG. 61 is a diagram of a portion of the “bias-tee” configuration ofFIG. 60, in accordance with embodiments of the present invention.

FIG. 62 is a side cross sectional view of another embodiment of nanoporememory chip having two inductors, one on each of the top Add chambers,in accordance with embodiments of the present invention.

FIG. 63 is a side cross sectional view of another embodiment of nanoporememory chip having one inductor one of the top Add chambers, inaccordance with embodiments of the present invention.

FIG. 63A is an equivalent circuit of a plurality of parallelnanopore-polymer resonators and signal processing having a single commoninductor connected to the top electrodes of the resonator and fixedcapacitance in each resonator cell, in accordance with embodiments ofthe present invention.

FIG. 63B is a side cross sectional view of another embodiment ofnanopore memory chip having the configuration of FIG. 63A, in accordancewith embodiments of the present invention.

FIG. 64 is a side cross sectional view of another embodiment of nanoporememory chip having an inductor on the bottom of the deblock chamber, inaccordance with embodiments of the present invention.

FIG. 64A is an equivalent circuit diagram of a “bias-tee” configurationto connect both AC and DC signals for the configuration of FIG. 64, inaccordance with embodiments of the present invention.

FIG. 64B is an equivalent circuit of a plurality of parallelnanopore-polymer resonators and signal processing having a single commoninductor and fixed capacitance in each resonator cell, in accordancewith embodiments of the present invention.

FIG. 64C is a side cross sectional view of another embodiment ofnanopore memory chip having the configuration of FIG. 64B, in accordancewith embodiments of the present invention.

FIG. 65 is a partial perspective view of a group of connected 3-chambercell nanopore devices having a transparent top and electrodes, inaccordance with embodiments of the present invention.

FIG. 66 is a partial perspective view of an alternative embodiment of agroup of connected 3-chamber cell nanopore devices having a transparenttop and electrodes, in accordance with embodiments of the presentinvention.

FIG. 67 is a circuit block diagram of an array of nanopore cellsconnected as per FIG. 65, in accordance with embodiments of the presentinvention.

FIG. 68 is a block diagram of a read/write memory controller and ananopore memory chip, in accordance with embodiments of the presentinvention.

FIG. 68A is a block diagram of a computer system, in accordance withembodiments of the present invention.

FIG. 69 is a table and graphs of memory add cycles and steering voltagesneeded to perform the cycles, in accordance with embodiments of thepresent invention.

FIG. 70 is a graph and data map showing how memory is populated forvarious data inputs using alternating write cycles, in accordance withembodiments of the present invention.

FIG. 70A is a flowchart of controller logic for performing the writecycles shown in FIG. 70, in accordance with embodiments of the presentinvention.

FIG. 70B is a table showing steps for writing “1” and “0” with thenanopore chip configured as shown in FIG. 66, in accordance withembodiments of the present invention.

FIG. 71 shows three different data format listings of the bits on amemory string, in accordance with embodiments of the present invention.

FIG. 72 shows a data format listing of the bits on a memory string foreach cell in a row, in accordance with embodiments of the presentinvention.

FIG. 73 shows an alternative data format listing of the bits on a memorystring for each cell in a row, in accordance with embodiments of thepresent invention.

FIG. 74 shows an alternative parallel data storage format listing of thebits on a memory string for cells in a row, in accordance withembodiments of the present invention.

FIG. 75 is a block diagram showing a nanopore memory system showing aread/write memory controller and an instrument for fluidics/reagents, inaccordance with embodiments of the present invention.

FIG. 76 is an gel electrophoresis preparation, showingtopoisomerase-mediated addition of oligonucleotide cassettes (“bits”) toa DNA molecule.

FIG. 77 depicts the DNA origami molecule of Example 7.

DETAILED DESCRIPTION

The following description of the preferred embodiment(s) is merelyexemplary in nature and is in no way intended to limit the invention,its application, or uses.

As used throughout, ranges are used as shorthand for describing each andevery value that is within the range. Any value within the range can beselected as the terminus of the range. In addition, all references citedherein are hereby incorporated by referenced in their entireties. In theevent of a conflict in a definition in the present disclosure and thatof a cited reference, the present disclosure controls.

Unless otherwise specified, all percentages and amounts expressed hereinand elsewhere in the specification should be understood to refer topercentages by weight. The amounts given are based on the active weightof the material.

“Nanochip” as used herein refers to a nanofluidic device, comprisingmultiple chambers containing fluid and optionally channels allowing forfluid flow, wherein the critical dimensions of the features of thenanochip, for example the width of the elements dividing the chambersfrom one another, are from one atom to 10 microns in thickness, e.g.,smaller than one micron, e.g. 0.01-1 micron. The flow of materials inthe nanochip may be regulated by electrodes. For example, as DNA and RNAare negatively charged, they will be drawn to a positively chargedelectrode. See, e.g., Gershow, M, et al., Recapturing and TrappingSingle Molecules with a Solid State Nanopore, Nat Nanotechnol. (2007)2(12): 775-779, incorporated herein by reference. The flow of fluids mayin some cases also be regulated by gate elements, and by flushing,injecting, and/or suctioning fluids into or out of the nanochip. Thesystem is capable of precise multiplexed analysis of nucleic acids(DNA/RNA). In certain embodiments, the nanochip can be made of a siliconmaterial, for example silicon dioxide or silicon nitride. Siliconnitride (e.g., Si₃N₄) is especially desirable for this purpose becauseit is chemically relatively inert and provides an effective barrieragainst diffusion of water and ions even when only a few nm thick.Silicon dioxide (as used in the examples herein) is also useful, becauseit is a good surface to chemically modify. Alternatively, in certainembodiments, the nanochip, may be made in whole or in part out ofmaterials which can form sheets as thin as a single molecule (sometimesreferred to as single layered materials), for example graphene, e.g., asdescribed in Heerema, S J, et al, Graphene nanodevices for DNAsequencing, Nature Nanotechnology (2016) 11: 127-136; Garaj S et al.,Graphene as a subnanometer trans-electrode membrane, Nature (2010) 467(7312), 190-193, the contents of each of which are incorporated hereinby reference, or a transition metal dichalcogenide, e.g., molybendumdisulfide (MoS₂) as described in Feng, et al., Identification of singlenucleotides in MoS ₂ nanopores, Nat Nanotechnol. (2015)10(12):1070-1076, the contents of which are incorporated herein byreference, or boron nitride, as described in Gilbert, et al. Fabricationof Atomically Precise Nanopores in Hexagonal Boron Nitride, eprintarXiv:1702.01220 (2017).

In some embodiments, the nanochip comprises such a single layeredmaterial which is relatively stiff and inert, e.g., at least as inertand stiff as graphene, such as MoS₂. Single layered materials may, forexample be used as all or part of the membrane comprising the nanopore.The nanochip may be lined in parts with metal, for example the walls maybe layered (e.g. metal-silicon nitride-metal), and the metal can then beconfigured to provide a controllable pair of electrodes near thenanopore, so that the nucleic acid can be moved back and forth throughthe nanopore by electromotive force, and also can be sequenced bymeasuring the change in electric potential as the nucleic acid passesthrough the nanopore.

Nanochip nanofluidic devices for sequencing DNA are generally known, forexample as described in Li, J., et al, Solid-state nanopore fordetecting individual biopolymers, Methods Mol Biol. (2009)544:81-93;Smeets R M, et al. Noise in solid-state nanopores, PNAS(2008)105(2):417-21; Venta K, et al., Differentiation of short,single-stranded DNA homopolymers in solid-state nanopores, ACS Nano.(2013)7(5):4629-36; Briggs K, et al. Automated fabrication of 2-nmsolid-state nanopores for nucleic acid analysis, Small(2014)10(10):2077-86; and Chen Z, DNA translocation through an array ofkinked nanopores, Nat Mater. (2010)9(8):667-75; the entire contents ofeach of which are incorporated herein by reference, e.g. for theirteachings on the design and manufacture of nanochips comprisingnanopores.

“Nanopore” as used herein is pore having a diameter of less than 1micron, e.g., 2-20 nm diameter, for example on the order of 2-5 nm.Single stranded DNA can pass through a 2 nm nanopore; single or doublestranded DNA can pass through a 4 nm nanopore. Having a very smallnanopore, e.g., 2-5 nm, allows the DNA to pass through, but not thelarger protein enzymes, thereby allowing for controlled synthesis of theDNA (or other charged polymer). Where larger nanopores (or smallerprotein enzymes) are used, the protein enzyme may be conjugated to asubstrate that will prevent it from passing though the nanopore, e.g. toa larger molecule, such as a larger protein, to a bead, or to a surfacein the chamber. Different types of nanopores are known. For example,biological nanopores are formed by assembly of a pore-forming protein ina membrane such as a lipid bilayer. For example, α-hemolysin and similarprotein pores are found naturally in cell membranes, where they act aschannels for ions or molecules to be transported in and out of cells,and such proteins can be repurposed as nanochannels. Solid-statenanopores are formed in synthetic materials such as silicon nitride orgraphene e.g., by configuring holes in the synthetic membrane, e.g.using feedback controlled low energy ion beam sculpting (IBS) or highenergy electron beam illumination. Hybrid nanopores can be made byembedding a pore-forming protein in synthetic material. Where there is ametal surface or electrode at either end or either side of the nanopore,a current flow across the nanopore may be established through thenanopore via an electrolyte media. Electrodes may be made of anyconductive material, for example silver, gold, platinum, copper,titanium dioxide, for example silver coated with silver chloride.

Methods for configuring a nanopore in a solid state, e.g., siliconnitride, membrane, are known. In one approach, a silicon substrate iscoated with the membrane material, e.g., silicon nitride, and theoverall configuration of the membrane is created using photolithographyand wet chemical etching, to provide silicon nitride membranes of thedesired size for incorporation into a nanochip, e.g., about 25×25microns. Initial 0.1 micron diameter holes or cavities are punched inthe silicon nitride membrane using a focused ion beam (FIB). Ion beamsculpting can configure the nanopore either by shrinking a larger pore,e.g., by ion beam induced lateral mass transport on the membranesurface, or by removing membrane material by ion beam sputtering layerby layer from the flat side of the membrane containing a cavity fromopposing sides, so that when the cavity is ultimately reached, there isa sharp-edged nanopore. The ion beam exposure is extinguished then theion current transmitted through the pore is appropriate for the desiredpore size. See, e.g. Li, J., et al., Solid-state nanopore for detectingindividual biopolymers, Methods Mol Biol. (2009)544:81-93.Alternatively, the nanopores can be configured using high energy(200-300 keV) electron beam illumination in a TEM. Using semiconductorprocessing techniques, e-beam lithography, reactive-ion etching of SiO2mask layers, and anisotropic KOH etching of Si, pyramidal 20×20 nm andlarger pores are made in a 40 nm thick membrane. The electron beam in aTEM is used to shrink the larger 20 nm pores to smaller ones. The TEMallows the shrinking process to be observed in real-time. Using athinner membrane (e.g., <10 nm thick) nanopores can be drilled with ahigh energy focused electron beam in a TEM. See, generally, Storm A J,et al. Fabrication of solid-state nanopores with single-nanometerprecision. Nature Materials (2003) 2:537-540; Storm A J, et al.Translocation of double-stranded DNA through a silicon oxide nanopore.Phys. Rev. E (2005)71:051903; Heng J B, et al. Sizing DNA Using aNanometer-Diameter Pore. Biophys. J (2004) 87(4):2905-11; the contentsof each of which are incorporated herein by reference.

In other embodiments, the nanopores are made using dielectric breakdown,using a relatively high voltage potential across the membrane, whereinthe voltage is raised until current is detected, e.g., as described inKwok, et al., “Nanopore Fabrication by Controlled Dielectric Breakdown,”PLOS ONE (2014) 9(3): e92880, the contents of which are incorporatedherein by reference.

Using these techniques, and depending of course on the exact techniqueused and the thickness and exact composition of the membrane, theoverall shape of the nanopore in a solid material such a silicon nitridemay roughly resemble two funnels with their apexes coming together atthe narrowest point, i.e., the actual nanopore. Such a double cone shapeis conducive to steering the polymer through the nanopore and back.Imaging techniques, for example atomic force microscopy (AFM) ortransmission electron microscopy (TEM), particularly TEM, can be used toverify and measure the size, location and configuration of thenanomembranes, the FIB holes or cavities, and the final nanopores.

In some embodiments, one end of the polymer, e.g., DNA, is tethered nearthe nanopore or on the inner wall of the funnel leading to the nanopore.Since the polymer approaches the nanopore initially by diffusion, thenis driven by the electrical gradient, the gradient-driven motion ismaximized and the diffusive motion minimized, and speed and efficiencythereby enhanced, if one end of the polymer is tethered close to thenanopore. See, e.g. Wanunu M, Electrostatic focusing of unlabelled DNAinto nanoscale pores using a salt gradient, Nat Nanotechnol. (2010)5(2):160-5; Gershow M., Recapturing and trapping single molecules with asolid-state nanopore. Nat Nanotechnol. (2007) 2(12):775-9; Gershow, M.,Recapturing and Trapping Single Molecules with a Solid State Nanopore.Nat Nanotechnol. (2007) 2(12): 775-779.

In one embodiment, one end of the polymer, e.g., DNA, is attached to abead and the polymer is driven through the pore. Attachment to the beadwill stop the polymer from moving all the way through the nanopore onthe opposite side of the dividing membrane in an adjacent chamber. Thecurrent is then turned off, and the polymer, e.g., DNA, attaches to thesurface adjacent to the nanopore in a chamber on the other side of thedividing membrane. For example, in one embodiment, one end of ssDNA iscovalently attached to a 50 nm bead, and the other end is biotinylated.Streptavidin is bound to the area at the desired point of attachment inthe chamber on the other side of the dividing membrane. The DNA ispulled through the nanopore by an electrical potential, and the biotinattaches to the streptavidin. The attachments to the bead and/or thesurface adjacent to the nanopore can be either covalent bonds or strongnoncovalent bonds (like the biotin-streptavidin bond). The bead is thencut off with an enzyme and flushed away. In some embodiments, the singlestranded DNA is cleaved with a restriction enzyme which cleaves singlestranded DNA, e.g., as described in K. Nishigaki, Type II restrictionendonucleases cleave single-stranded DNAs in general. Nucleic Acids Res.(1985) 13(16): 5747-5760, incorporated herein by reference. In otherembodiments, a complementary oligonucleotide is provided to make adouble-stranded restriction site, which can then be cleaved with thecorresponding restriction enzyme.

As the polymer passes through the nanopore, the change in electricpotential, capacitance or current across the nanopore caused by thepartial blockage of the nanopore as the polymer passes through can bedetected and used to identify the sequence of monomers in the polymer,as the different monomers can be distinguished by their different sizesand electrostatic potentials.

The use of nanochips comprising nanopores in a method of DNAfabrication, as described herein, is not disclosed in the art, but suchchips are well known and commercially available for rapid sequencing ofDNA. For example, the MinION (Oxford Nanopore Technologies, Oxford, UK)is small and can be attached to a laptop computer. As a single strand ofDNA passes through a protein nanopore at 30 bases per second, the MinIONmeasures the electrical current. The DNA strands in the pore disruptsthe ionic flow, resulting in changes in current corresponding to thenucleotides in the sequence. Mikheyev, A S, et al. A first look at theOxford Nanopore MinION sequencer, Mol. Ecol. Resour. (2014)14,1097-1102. While the accuracy of the MinION is poor, requiring repeatedresequencing, the speed and accuracy of the sequencing using thenanochips of the present invention can be greatly improved if the DNAbeing read contains only two easily distinguishable bases, e.g. A and C.

The membrane comprising the nanopores may, in some embodiments, have atrilayer configuration, with a metal surface on either side of aninsulating core material, e.g. a silicon nitride membrane. In thisembodiment, the metal surfaces are configured, e.g., by lithographicmeans, to provide a microcircuit with paired electrodes, one at each endof each nanopore, e.g., such that a current flows across the nanoporemay be established between the electrodes and through the nanopore viaan electrolyte media, which current can draw the polymer through thenanopore and by reversing the polarity, can draw it back. As the polymerpasses through the nanopore, the electrodes can measure the change inelectric potential across the nanopore so as to identify the sequence ofmonomers in the polymer.

In some embodiments, the sequence of the polymer is designed to storedata. In some embodiments, the data is stored in a binary code (1's and0's). In some embodiments each base corresponded to a 1 or 0. In otherembodiments, an easily recognized sequence of two or more basescorresponds to a 1 and another easily recognized sequence of two or morebases corresponds to a 0. In other embodiments, the data is can bestored in a ternary, quaternary or other code. In a particularembodiment, the polymer is DNA, for example single stranded DNA, whereinthe DNA contains only two base types and does not contain any basescapable of self-hybridizing, e.g., wherein the DNA comprises adeninesand guanines, adenines and cytosines, thymidines and guanines, orthymidines and cytosines. In some embodiments, the two bases may beinterspersed with one or more additional bases, for example A and C maycontain a T to “punctuate” the sequence, e.g., by indicating a break ina coding sequence, at a frequency that does not result in significantself-hybridization. In other embodiments, e.g., where the nucleic acidis double stranded, some or all available bases may be employed.

The nucleotide bases may be natural or may in some embodiments consistof or include nonnatural bases, e.g. as described in Malyshev, D. et al.“A semi-synthetic organism with an expanded genetic alphabet”, Nature(2014) 509: 385-388, incorporated herein by reference.

In one embodiment, the data is stored by addition of single monomers,e.g., single nucleotides in the case of DNA, to the polymer. In oneembodiment, the polymer is DNA and the monomers are adenine (A) andcytosine (C) residues. A and C residues have an advantage because (i) Aand C have a large size difference, so differentiation through thenanopore should be facilitated, (ii) A and C do not pair with oneanother so do not form significant secondary structure which couldcomplicate interpretation of the nanopore signal, and (iii) for the samereason, G's are less preferred as they are know to form guanine tetrads.Nucleotides are added by terminal transferase (or polynucleotidephosphorylase), but the nucleotides are 3′-blocked so that only a singlenucleotide is added at a time. The block is removed prior to addition ofthe next nucleotide.

In some embodiments, the DNA is left in the nanochip. In otherembodiments, it is removed, and optionally converted to double strandedDNA and/or optionally converted to crystalline form, e.g. to enhancelong term stability. In still other embodiments, DNA can be amplifiedand the amplified DNA removed for long term storage, while the originaltemplate DNA, for example DNA bound to the wall of a chamber in thenanochip, can be left in the nanochip, where it can be read and/or usedas a template to make additional DNA.

In some embodiments, the DNA or other polymer is anchored to a surfaceproximate to the nanopore during synthesis. For example, in oneembodiment, single stranded DNA molecules are each attached at the 5′end to a surface proximate to a nanopore, wherein the current at eachnanopore can be independently regulated by electrodes for that nanopore,so that the 3′end of the DNA molecule can be pulled forward through thenanopore from a retaining chamber into a flow chamber containing a flowof 3′-protected dNTPs together with a polymerase or terminal transferaseenzyme to add a 3′-protected dNTPs, or retained in the retaining chamberwhere the nanopore excludes the enzyme, so that the dNTP is not added.See, e.g., depictions at FIGS. 12-16 and also FIGS. 18 and 19. In otherembodiments, single stranded DNA is built by addition to the 5′ end(with the 3′ end attached), using topoisomerase, as described more fullybelow. By controlling whether or not each DNA molecule participates ineach cycle, the sequence of each DNA molecule can be preciselycontrolled, e.g., as follows:

Flow Step Chamber Nanopore 1 Nanopore 2 0 Retain Retain 1 flow ‘A’ 2Forward into flow retain chamber ‘A’ gets added 3 Reverse back intoretain resting chamber; oligo is deprotected 4 Flush with retain retainbuffer 5 flow ‘C’ forward forward ‘C’ gets added ‘C’ gets added 6reverse reverse oligo is deprotected oligo is deprotected 7 retainforward ‘C’ gets added 8 retain reverse oligo is deprotected 9 Flushwith retain retain buffer Flow A = 3′-protected dATP Flow C =3′-protected dCTPNanopore 1 and Nanopore 2 in this schematic are associated withdifferent DNA strands and the positions of which (in or out of the flowchambers) are separately controllable. The DNA can be deprotected eitherby a specific enzyme in the retaining chamber, or by changing the flowin the flow chamber to provide deprotection by enzymatic, chemical,light-catalyzed or other means. In one embodiment, the deblockingagent(s) flow between cycles of Flow A and Flow C, e.g., when the flowchamber is being washed with buffer, so that the deblocking agent doesnot deprotect the nucleotide building blocks. In other embodiments, thedeprotecting agent(s) are too bulky to cross to the flow chamber via thenanopores.

The end result in the foregoing example would be that an A and a C wereadded to the DNA at Nanopore 1, and a C and a C were added to the DNA atNanopore 2.

In another embodiment, the chamber configuration is similar, but withdouble stranded DNA anchored to the surface proximate to a nanopore, andoligonucleotide fragments, for example of two or more types, eachcorresponding to a binary code, are added sequentially, e.g., usingsite-specific recombinases, i.e., enzymes that spontaneously recognizeand cleave at least one strand of a double strand of nucleic acidswithin a sequence segment known as the site-specific recombinationsequence, for example using topoisomerase-charged oligonucleotides asdescribed below.

In certain embodiments, it may be desirable to keep the electricallycharged polymer, e.g., DNA, in a condensed state subsequent tosynthesis. There are several reasons for this:

-   -   the polymer should be more stable in this form,    -   condensing the polymer will keep down crowding and allow use of        longer polymers in small volumes,    -   orderly condensation can reduce potential that the polymer will        form knots or tangles,    -   if any of the chambers are interconnected it will help keep the        polymer from getting so long that it goes through a different        pore than it is supposed to when current is applied,    -   condensation will help keep polymer away from the electrodes,        where electrochemistry could damage the polymer.        A human cell is about 10 microns but contains 8 billion base        pairs of DNA. Stretched out it would be over a meter long. The        DNA fits into the cell because it is wound around histone        proteins. In certain embodiments, histones or similar proteins        provide a similar function in the nanochips of the invention. In        some embodiments, the interior surfaces of the nanochips are        slightly positively charged so that electrically charged        polymer, e.g., DNA tends to stick weakly to them.

In certain embodiments, the charged polymer, e.g., single or doublestranded DNA, bound to a surface proximate to a nanopore. This can beaccomplished in various ways. Generally, the polymer is localized to thenanopore by attaching the polymer to a relatively bulky structure (e.g.a bead, a protein, or a DNA origami structure (described below), havinga diameter too large to fit through the nanopore, e.g., >10 nm, e.g.,about 20-50 nm), pulling the charged polymer through the nanopore usingcurrent, anchoring the end of the polymer distal to the bulky structureto the surface adjacent to the nanopore, for example wherein the surfaceis modified to accept a linker group attached to the distal end of thepolymer strand, thereby attaching the polymer strand, and cleaving offthe bulky structure.

The step of anchoring the end of the polymer distal to the bulkystructure to the surface adjacent to the nanopore, can be accomplishedin various ways. In one embodiment, the polymer is a single strandedDNA, and there are pre-attached DNA strands (about 50 bp) which arecomplementary to part of the single stranded DNA, so that the singlestranded DNA and the pre-attached DNA strands can join via base pairing.If the pairing is strong enough, it will be sufficient to keep the DNAanchored even while being manipulated. An advantage of this method ofattachment is that it allows the DNA to be removed from the nanoporechip if desired for long term storage of the DNA. Alternatively, thestrand is attached to the surface covalently, either using conjugationchemistry, e.g., streptavidin-biotin conjugation as described in Example1 below, or ‘click’ chemistry (see Kolb, et al. Angew. Chem. Int. Ed.(2001)40: 2004-2021, incorporated herein by reference, and/or usingenzymatic attachment, for example by pre-attaching oligos covalently tothe distal surface, and then using DNA ligase to connect them.

Once the distal end of the strand is attached to the surface adjacent tothe nanopore, the bulky structure is cleaved off, e.g., using anendonuclease which cleaves at a restriction site near the bulkystructure.

The bulky structure may be a bead, a bulky molecule, e.g., a proteinwhich is reversibly bound to a DNA strand, or a DNA origami structure.DNA origami involves the use of base pairing to create three dimensionalDNA structures. DNA origami techniques are generally described in Bell,et al, Nano Lett. (2012)12: 512-517, incorporated herein by reference.For example, in the current invention, DNA origami can be used to attachthe single DNA molecule to a surface adjacent to the nanopore. In oneembodiment, the structure is a ‘honey comb cube’, e.g., about 20 nm oneach side. This prevents this part of the DNA from going through thenanopore (just like in the attached paper). There is a long strand ofDNA (single or double stranded) attached to the origami structure. TheDNA strand goes through the nanopore, until the origami cube meets thenanopore and blocks further progress. The current is then turned off andthe strand is attached to the surface adjacent to the nanopore.

In another embodiment, the electrically charged polymer, e.g., DNA, withthe origami structure is in the middle chamber of a three chamberconfiguration. The origami will keep the DNA from completely enteringthe other 2 chambers (or other one chamber in the 2 chamber example).Thus, in this example the polymer doesn't need to be anchored to thesurface. This reduces the risk that the polymer will knot up and avoidsthe need for the step of binding one end of the polymer to the surfaceand cleaving off the bulky portion at the other end. The volume of thechamber with the origami should be kept as small as practical so thatthe polymer stays relatively close to the pore, which will help ensurethat it translocates quickly when current is applied. It should be notedthat while the middle chamber containing the origami portion of thepolymer can't be interconnected with other middle chambers (or else thedifferent polymers will get mixed up), the other chambers (or sets ofchambers in the 3 chamber example) can be interconnected. These otherchambers can have larger volumes if desired, as the polymer willnecessarily be close to the pore (some of it will be in the pore infact) when the DNA is moved to that chamber.

In some embodiments, the device comprises three in-line chambers,wherein the addition chambers are contiguous to allow for flow, and havecommon electrodes, while the ‘deprotect’ chambers are fluidicallyisolated except for the flow through the nanopore and have uniqueelectrodes.

In other embodiments, the DNA or other charged polymer is not anchoredbut can move between synthesis chamber(s) and deprotection chamber(s),under control of electrodes in the chambers, while the polymerase andthe deprotecting agents are restricted from movement between chambersbecause they are too bulky to pass through the nanopores connecting thechambers and/or are anchored to a surface in a chamber. See, e.g., FIGS.1-9 and 16-17.

The current needed to move the charged polymer through the nanoporedepends on, e.g., the nature of the polymer, the size of the nanopore,the material of the membrane containing the nanopore, and the saltconcentrations, and so will be optimized to the particular system asrequired. In the case of DNA as used in the examples herein, examples ofvoltage and current would be, e.g., 50-500 mV, typically 100-200 mV, and1-10 nA, e.g., about 4 nA, with salt concentrations on the order of 100mM to 1M.

The movement of charged polymer, e.g., DNA, through the nanopore isnormally very rapid, e.g., 1 to 5 μs per base, so on the order of onemillion bases per second (1 MHz, if we adopt the nomenclature offrequency), which presents challenges for getting an accurate readingdistinct from the noise in the system. Using current methods, either (i)a nucleotide needed to be repeated in a sequence, e.g., ca. 100 timessuccessively, in order to produce a measurable characteristic change, or(ii) using protein pores, such as Alpha hemolysin (αHL) or Mycobacteriumsmegmatis porin A (MspA), which provide a relatively long pore withpotential for multiple reads as the base moves through the polymer, andin some cases, can be adapted to provide a controlled feed of DNAthrough the pore one base at a time, in some cases using an exonucleaseto cleave each base as it passes through. Various approaches arepossible, e.g.,

-   -   slowing down the speed of the polymer, from ca. 1 MHz to ca.        100-200 Hz, for example using a medium comprising an        electrorheological fluid in which becomes more viscous when a        voltage is applied, thereby slowing down the speed of the        polymer through the nanopore, or a plasmonic fluid system,        wherein the viscosity of the medium can be controlled by light;        or a molecular motor or ratchet;    -   providing a sequence in the polymer, e.g., in single stranded        DNA, which will form a bulky secondary structure, e.g., a        “hairpin”, “hammerhead”, or “dumbbell” configuration, which will        have to be linearized in order to fit through the nanopore,        thereby making the information less dense and providing a signal        having a longer duration;    -   providing many reads of the same sequence, e.g., by using        rapidly alternating current, allowing for many reads of the same        sequence frame, and combined with brief bursts of direct current        to pull the molecule to the next sequence frame, by reading the        entire sequence multiple times, or by reading multiple identical        sequences in parallel, in each case collating the reads to        provide a consensus read that amplifies the signal;    -   measuring an impedance change in a high frequency signal induced        by a change in capacitance as monomers (e.g., nucleotides) pass        through the nanopore, rather than measuring changes in current        flow or resistance directly;    -   enhancing the differences in current, resistance or capacitance        between different bases, e.g., by using non-natural bases which        have a greater difference in size or are otherwise modified to        give different signals, or by forming larger secondary        structures within the DNA, such as a “hairpin”, “hammerhead”, or        “dumbbell” configuration, which provide an enhanced signal        because of their larger size;    -   using an optical reading system, for example using an integrated        optical antenna adjacent to the nanopore, which acts as an        optical transducer (or optical signal enhancer) to complement or        replace standard ion current measurements, e.g., as described in        Nam, et al., “Graphene Nanopore with a Self-Integrated Optical        Antenna”, Nano Lett. (2014)14: 5584-5589, the contents of which        are incorporated herein by reference. In some embodiments, the        monomers, e.g. DNA nucleotides, are labeled with fluorescent        dyes so that each different monomer fluoresces at a signature        intensity as it passes through the junction of the nanopore and        its optical antenna. In some embodiments, a solid-state nanopore        strips off fluorescent labels, leading to a series of detectable        photon bursts, as the polymer passes through the nanopore at        high speed, e.g. as described in McNally et al., “Optical        recognition of converted DNA nucleotides for single molecule DNA        sequencing using nanopore arrays”, Nano Lett. (2010)10(6):        2237-2244, and Meller A., “Towards Optical DNA Sequencing Using        Nanopore Arrays”, J Biomol Tech. (2011) 22(Suppl): S8-S9, the        contents of each of which are incorporated herein by reference.

In one embodiment, the charged polymer is a nucleic acid, e.g., singlestranded DNA, wherein the sequences provide a secondary structure. Bell,et al., Nat Nanotechnol. (2016)11(7):645-51, incorporated herein byreference, describes using a relatively short sequence of dumbbellconfigurations detectable in a solid state nanopore format, to labelantigens in an immunoassay. The nanopores used in Bell, et al. wererelatively large, so the entire dumbbell structure could pass throughthe pore, but using nanopores smaller than the diameter of the dumbbellconfiguration, the DNA will “unzip” and become linearized. More complexconfigurations can be used, e.g. wherein each bit corresponds to asequence similar to a tRNA (see, e.g., Henley, et al. Nano Lett.(2016)16: 138-144, incorporated herein by reference). Thus the inventionprovides charged polymers, e.g. single stranded DNA, having at least twotypes secondary structure, wherein the secondary structure encode data(e.g. binary data, wherein one secondary structure type is a 1 and asecond is a 0). In other embodiments, secondary structures are used toslow down the passage of the DNA through the nanopore or to providebreaks in the sequence, to facilitate reading of the sequence.

In another embodiment, the invention utilizes a DNA molecule comprisinga series of at least two different DNA motifs, wherein each motifspecifically binds to a particular ligand, for example a gene regulatoryprotein for double stranded DNA or a tRNA for single stranded DNA,wherein the at least two different DNA motifs encode information, e.g.in a binary code, wherein one motif is a 1 and a second is a 0, e.g.,wherein the ligand enhances the signal difference (e.g. change incurrent or capacitance) across the nanopore as the DNA passes throughthe nanopore.

As discussed above, when different monomers pass through the nanopore,they affect the current flow through the nanopore, primarily byphysically blocking the nanopore and changing the conductance across thenanopore. In existing nanopore systems, this change in current ismeasured directly. The problem with current reading systems is thatthere is considerable noise in the system, and in the case of DNA, forexample, when measuring current fluctuations as different nucleotideunits pass through the nanopore, a relatively long integration time, onthe order of one hundredth of a second, is needed to accurately detectdifferences between different monomers, e.g., between different bases.Recently, it has been shown that changes in impedance and capacitancecan be useful to study cells and biological systems, despite thepotential for complex interactions with salts and biological molecules.For example, Laborde, et al. Nat Nano. (2015)10(9):791-5 (incorporatedherein by reference), demonstrates that high-frequency impedancespectroscopy can be used to detect small changes in capacitance underphysiological salt conditions and image microparticles and living cellsbeyond the Debye limit.

In one embodiment of the invention, therefore, we measure capacitivevariance rather than measuring current variance directly, for example,wherein the sequence of the charged polymer is identified by measuringthe phase change in a radiofrequency signal induced by change incapacitance as the monomers (e.g., nucleotides) pass through thenanopore.

Simply stated, capacitance exists in any circuit where there is a gapbetween one electrical conductor and another. While current variesdirectly with capacitance, it does not vary simultaneously withcapacitance. For example, if we were to plot the current and voltageover time in a capacitive circuit with an alternating electricalcurrent, we would see that while both current and voltage each form asine wave, the waves are out of phase. When there is a change incurrent, there is a change in capacitance, which is reflected in achange in the phase of the signal. A radiofrequency alternating currentprovides a signal with fixed frequency and amplitude, while the phase ofthe signal will vary with the capacitance of the circuit. In our system,we use a pulsating direct current rather than an alternating current(i.e., the voltage alternates between two values, but the voltage doesnot cross the “zero” line, such that polarity is maintained and oneelectrode remains positive and the other negative), so that the chargedpolymer can be drawn through the nanopore (towards the positiveelectrode in the case of DNA). When there is nothing in the nanopore,the capacitance has one value, which changes as the different monomersof the polymer pass through the nanopore. Suitable frequency ranges arein the radiofrequency range, e.g. 1 MHz to 1 GHz, e.g. 50-200 MHz, forexample about 100 MHz, e.g. below higher microwave frequencies thatcould cause significant dielectric heating of the medium. To reduce thepotential for interference, different frequencies can be applied atdifferent nanopores so that multiple nanopores can be measuredsimultaneously with a single radiofrequency input line.

Measuring impedance changes (due to, e.g. changes in capacitance) athigh frequencies increases the signal to noise available within acertain time span, as it reduces the effects of 1/f noise, or ‘pink’noise that is inherent in electronic measurement circuits. Using a highfrequency signal enhances the signal-to-noise ratio, as manymeasurements are made within a given time span, providing a more stablesignal which is readily distinguished from impedance changes due toenvironmental or device variation and fluctuation.

Applying these principles to the instant invention, the inventionprovides in one embodiment, a method of measuring an impedance change ina high frequency signal induced by a change in capacitance as monomers(e.g., nucleotides) pass through a nanopore, for example, a method ofreading a monomer sequence of a charged polymer comprising at least twodifferent types of monomers, for example a DNA molecule, comprisingapplying a radiofrequency pulsating direct current, e.g. at a frequencyof 1 MHz to 1 GHz, e.g. 50-200 MHz, for example about 100 MHz, across ananopore, wherein the pulsating direct current draws the charged polymerthrough the nanopore and the monomer sequence is read by measuring thecapacitive variance across the nanopore as the charged polymer goesthrough the nanopore.

For example, referring to FIGS. 48A, 48B, 48C, a nanopore-based cell4800 is shown having an upper (or top) chamber 4802 and a lower (orbottom) chamber 4804 and a membrane 4806, which separates the twochambers 4802, 4804. The membrane is made of a material as describedherein above. Also in the cell is a nanopore 4808 (or nanometer-sizedhole) through the membrane, having a shape and dimensions such at thatdescribed herein, which allows for fluid communication between thechambers 4802, 4804.

Inside the cell 4800, is a polymer molecule, e.g., a single-stranded DNAmolecule 4810 (or ssDNA), such as that described herein above. In thisexample, the DNA 4810 has three units or bases, an upper base 4812, amiddle base 4814, and a lower base 4816. Each of the bases 4812-4816 inthe DNA 4810, or a collection of bases, may be referred to as a “bit” ofinformation used to represent or store data, as also discussed herein.Any other polymer or DNA molecule (single or double-stranded) may beused if desired, as discussed herein above.

The chambers 4802, 4804, of the cell 4800 may be filled with a fluid,such as that describe herein, that allows the DNA 4810 to float and movebetween the chambers 4802,4804. The cell 4800 also has a pair ofelectrodes 4818, 4820, an upper (or top) electrode 4818 connected to aninput voltage Vin, and a lower (or bottom) electrode 4820 connected toground (or GND or 0 volts). In some embodiments, the lower electrode4820 may also be connected to a non-zero DC voltage, but may still be atAC (or rf or radio frequency) “ground” through use of an AC couplingcapacitor, connected to the electrode, having a capacitance value whichpasses the AC voltage (discussed more hereinafter). Voltage applied tothe electrodes 4818,4820 determines the movement of the DNA 4810 in thecell 4800. In particular, when the DNA strand 4810 is in the presence ofan electric field or voltage or charge difference, the DNA 4810 will beattracted to the positive charge or voltage, because the DNA molecule4810 has a net negative charge, as described hereinbefore with FIGS.1-5.

In this case, when the top electrode 4818 has a positive voltagerelative to the bottom electrode 4820 (shown here at ground or 0 volts),the DNA 4810 will move through the nanopore 4808 (if it was in the lowerchamber 4804) toward the top electrode 4818, and into the upper chamber4802. Conversely, when the top electrode 4818 has a negative voltagerelative to the bottom electrode 4820, the DNA 4810 will move throughthe nanopore 4808 toward the bottom electrode 4818, and into the lowerchamber 4804. FIGS. 48A, 48B and 48C shows the DNA 4810 moving throughthe nanopore 4808 from the upper chamber 4802 to the lower chamber 4804.

Referring to FIGS. 48A-48C, the cell 4800 (or the nanopore and DNAsystem) may be electrically modeled as an equivalent circuit diagram4830, shown as a capacitor C1 and resistor R1 connected in parallel. Inparticular, the top electrode 4818 sees a capacitance C1 and resistanceR1 to ground that is set by its local environment, where the capacitorC1 represents the capacitance of the cell 4800 as determined by theproperties of the two electrodes 4818,4820 (i.e., the capacitor plates)and the properties of dielectric material there-between, defined atleast by the fluid within the cell 4800 and the membrane 4806 with thenanopore 4808. The resistor R1 represents the DC resistance associatedwith the cell 4800, defined at least by the losses associated with thedielectric material of the cell described above, which appear as a DCleakage current between the two electrodes.

When the DNA 4810 passes through the nanopore 4808, both the cellcapacitance and resistance (or the cell impedance, Zcell) changes.Different DNA bases have different sizes, and thus have differenteffects on the capacitance and resistance, resulting in differentequivalent circuit models as illustrated in FIGS. 48A-48C. Inparticular, in FIG. 48A, the DNA 4810 is outside the nanopore 4808,resulting in a set of values C1,R1 (or Zcell1). In FIG. 48B, the base4814 of the DNA 4810 is in the nanopore, resulting in another set ofvalues C2,R2 (or Zcell). Similarly, in FIG. 48C, the base 4812 of theDNA 4810 is in the nanopore, resulting in another set of values C3,R3(or Zcell3).

Referring to FIGS. 49A and 49B, the capacitance C and resistance R ofthe cell 4800 (nanopore and DNA system) may be combined with an inductorL to create an “inductor-cell” or “cell-inductor” RLC resonant circuitor resonator or filter (or band-stop, or notch, or band-reject filter)as shown by the circuit 4900 (FIG. 49A), having a magnitude frequencyresponse shown by a graph 4952, and a phase frequency response shown bya graph 4954. The center or resonant frequency f_(res) of the circuit4900 is given by the equation:ω_(res)=2πf _(res)=Sqrt(1/LC)×Sqrt(1−(L/C)/R ²)  Eq.1where C and R are the capacitance and resistance of the cell,respectively, at a given time, and L is the value of the inductor (whichis a constant with respect to the position of the DNA in the cell).There may also be an equivalent coil resistor (not shown) in series withthe inductor L, indicative of the DC resistance of the inductor coilwinding. However, if the coil resistance is negligible, it need not beshown in the circuit diagram or in the resonant frequency f_(res)equation.

When the value of the cell resistance R is large, Eq.1 becomes:ω_(res)=2πf _(res)=Sqrt(1/LC)  Eq.2

Referring to FIG. 49B, the magnitude response 4952 of the resonantcircuit 4900 (for a given value of C and R) has maximum attenuation(minimum impedance) at the resonant frequency f_(res), and a steepmagnitude attenuation response over a narrow frequency band (or stopband) Δf_(stp) around the resonant frequency f_(res), which is thefrequency range over which the magnitude response (Vout/Vin) is below astandard threshold, e.g., 3 dB (or 20 Log[SQRT(2)]). For all otherfrequencies, the output magnitude is substantially constant andnon-attenuating. The corresponding phase shift response curve 4954 ofthe resonant circuit 4900 has a phase shift of 45 degrees at theresonant frequency f_(res) (when the reactance or imaginary portion ofthe complex impedance is equal to zero), and has a steep phase responseover the narrow stop band Δf_(stp) on each side of the resonantfrequency f_(res), such that at frequencies above f_(res) and outsidethe band Δf_(stp), the phase shift is at or near 180 deg., and atfrequencies below f_(res) and outside the band Δf_(stp), the phase shiftis at or near 0 deg. Otherwise, the phase response output issubstantially constant and non-shifting over all other frequencies.

Referring to FIG. 50, family of resonant frequency response magnitudecurves 5002 and phase curves 5003 of the resonant circuit (or filter)are shown in response to DNA (or other polymer or molecule havingvarying sizes along its length) passing through a nanopore and changingthe capacitance (or impedance) measured to ground (e.g., 0 volts), andthereby changing the resonant frequency f_(res), as shown by themagnitude response curves 5010-5018 and the corresponding phase responsecurves 5020-5028. In particular, as shown by Eq.2 above, increasing thecapacitance measured to ground, decreases the resonant frequencyf_(res). Also, increasing the DNA “base” size (blocking the nanoporemore), may increase the cell capacitance (depending on the dielectricconstant of the DNA), which would decrease the resonant frequencyf_(res). Conversely, decreasing the DNA base size (un-blocking thenanopore more), may decrease the cell capacitance (depending on thedielectric constant of the DNA), which would increase the resonantfrequency f_(res). Using standard DNA bases (G,C,A,T), the size orderwould be: G (largest), A, T, C (smallest). Accordingly, presuming thedielectric constant of the DNA changes appropriately with the DNA bases,when DNA is in the nanopore, f_(res) would be lowest when the largestbase, e.g., Base G, is in the nanopore and f_(res) would be the highestwhen the smallest base, e.g., Base C, is in the nanopore. Also, when thenanopore is open (unblocked), i.e., no DNA or polymer in the nanopore,f_(res) would be the highest frequency. This range or band of resonantfrequencies is shown as Δf_(res) in FIG. 50. Also, for the resonatorresponse curves 5010-5018 for magnitude and 5020-5028 for phase shown inFIG. 50, the overall bandwidth of the resonator (over which magnitudesand phase are materially affected by the resonator) is shown in FIG. 50as Δf_(BW).

In addition, the overall resonant frequency band Δf_(res) for a givencell-inductor resonant circuit (or resonator or filter), presuming allother cell conditions affecting impedance remained fixed, would have amaximum resonant frequency f_(res-max) when the nanopore is open (orunblocked by the polymer), and a minimum resonant frequency f_(res-min)when the nanopore is closed (or blocked by the polymer), and the cellresonant frequency f_(res) is varied (or tuned or changed) by the sizeof the polymer in the nanopore at a given time, and thus may be referredto herein as a nanopore-polymer resonator (or NPR). Also, the overallbandwidth Δf_(BW) of the resonator (over which magnitudes and phase arematerially affected by the resonator) would similarly be determinedbased on the resonant frequency band Δf_(res).

When there is an AC voltage applied to the resonator and the outputvoltage signal (Vout) of the resonator is observed (or monitored) at afixed frequency f_(probe), e.g., near the center of the resonantfrequency range for the DNA bases shown, shown as a dashed vertical line5004, not necessarily aligned with one of the center (or resonant)frequencies f_(res), the resonator provides four different possibleoutput signals (or magnitude attenuation or phase shift amounts) at thatfrequency f_(probe), depending on the specific DNA base (size) passingthrough the nanopore, and a fifth output voltage state when the DNA isnot in the nanopore (“open pore” condition). These five output signalsare shown by where the family of response curves 5002,5003 intersectwith the line 5004 corresponding to the monitor frequency magnitudesV1-V5, and f_(probe), i.e., phases Ph1-Ph5.

Alternatively, if the output voltage is monitored at a differentfrequency f_(probe2), e.g., near the resonant frequency of the lowestfrequency response curve 5010, shown as a dashed vertical line 5006,which is, e.g., at a lower frequency than f_(probe), the output voltageat the frequency f_(probe2) would be five magnitude output voltagesV6-V10 (different from the output voltages V1-V5 seen at f_(probe)) andfive output phase shifts Ph6-Ph10 (different from the output voltagesPh1-Ph5 seen at f_(probe)). In particular, these different five outputsignals are shown by where the family of response curves 5002, 5004intersect with the line 5006 corresponding to the monitor frequencyf_(probe2), i.e., magnitudes V6-V10, and phases Ph6-Ph10. The values ofV1-V10 and Ph1-Ph10 are arbitrary designations and are used foridentification or labeling purposes only. The value or location for theprobe or monitoring frequency f_(probe), f_(probe2) may be set based onthe desired response values, as described below. As shown below, usingvalues for the probe or monitoring frequencies in the range of resonantfrequencies seen when the polymer is in the nanopore, may provide auseful range of output values. Other measurement or probe frequenciesmay be used if desired provided it meets the desired functional andperformance requirements.

In particular, if the four bases were to pass through the nanoporesequentially in size order (i.e., G, A, T, C), when the largest base(e.g., Base G) passes through the nanopore, the resonant frequencyf_(res) will be the lowest (capacitance highest), and the correspondingresponse is shown by the (far left) magnitude and phase curves 5010,5020, respectively. In that case, the output voltage at the frequencyf_(probe) where the curves 5010, 5020 intersect with the line 5004 wouldcorrespond to an output voltage V2 and output phase Ph1, respectively.Alternatively, if the output voltage is monitored at the monitorfrequency f_(probe2), the output voltage at the frequency f_(probe2)where the curves 5010, 5020 intersect with the line 5006 wouldcorrespond to an output voltage V10 and output phase Ph6, respectively.

Similarly, when the next smaller base (e.g., Base A) passes through thenanopore, the resonant frequency f_(res) will be a little higher thanthat of the previous base, and the response is shown by the magnitudeand phase curves 5012, 5022, respectively. In that case, the outputvoltage at the frequency f_(probe) where the curves 5012, 5022 intersectwith the line 5004 would correspond to an output voltage V5 and outputphase Ph2, respectively. Alternatively, if the output voltage ismonitored at the monitor frequency f_(probe2), the output voltage at thefrequency f_(probe2) where the curves 5012, 5022 intersect with the line5006 would correspond to an output voltage V9 and output phase Ph7,respectively.

Similarly, when the next smaller base (e.g., Base T) passes through thenanopore, the resonant frequency f_(res) will be a little higher thanthat of the previous base, and the response is shown by the magnitudeand phase curves 5014, 5024, respectively. In that case, the outputvoltage at the frequency f_(probe) where the curves 5014, 5024 intersectwith the line 5004, would correspond to an output voltage V4 and outputphase Ph3, respectively. Alternatively, if the output voltage ismonitored at the monitor frequency f_(probe2), the output voltage at thefrequency f_(probe2) where the curves 5014, 5024 intersect with the line5006 would correspond to an output voltage V8 and output phase Ph8,respectively.

When the smallest base (e.g., Base C) passes through the nanopore, theresonant frequency f_(res) will be a little higher than that of theprevious base, and the response is shown by the magnitude and phasecurves 5016, 5026, respectively. In that case, the output voltage at thefrequency f_(probe) where the curves 5016, 5026 intersect with the line5004 would correspond to an output voltage V3 and output phase Ph4,respectively. Alternatively, if the output voltage is monitored at themonitor frequency f_(probe2), the output voltage at the frequencyf_(probe2) where the curves 5016, 5026 intersect with the line 5006would correspond to an output voltage V7 and output phase Ph9,respectively.

Lastly, when there is no DNA in the nanopore, the resonant frequencyf_(res) will be the highest (capacitance lowest), and the response isshown by the magnitude and phase curves 5018, 5028, respectively. Inthat case, the output voltage at the frequency f_(probe) where thecurves 5018, 5028 intersect with the line 5004, would correspond to anoutput voltage V1 and output phase Ph5, respectively. Alternatively, ifthe output voltage is monitored at the monitor frequency f_(probe2), theoutput voltage at the frequency f_(probe2) where the curves 5018, 5028intersect with the line 5006 would correspond to an output voltage V6and output phase Ph10, respectively.

Referring to FIG. 51, an overview example of a DNA-reading time sequence5100 in accordance with embodiments the present disclosure is shown forreading data stored in DNA using two-bits (e.g., two bases) in the DNAstrand 4810 via the “inductor-cell” resonator or “capacitive-resonance”DNA data reading technique of the present disclosure measured at a fixedprobe (or monitor) frequency f_(probe). The DNA-reading time sequencehas 5 time segments or stages T1-T5, shown as five columns 5102 to 5110,and the progression between time segments is shown by dashed lines 5111.For each of the time stages T1-T5, there is an image showing the DNA4810 (FIG. 48) and its location relative to the nanopore 4808 for thattime stage, there are also magnitude and phase response curves 5112-5120for the time stages T1-T5, respectively, showing the intersection of theresponse curve at the monitor frequency f_(probe) (corresponding to acurve from the family of curves shown in FIG. 50), and two output valuegraphs 5130, 5132 showing corresponding output signals (e.g., voltagevalues) for the magnitude and phase responses, respectively, at themonitor frequency f_(probe) across each of the time stages T1-T5. Thevalues V1-V5 and Ph1-PH5 for the graphs 5130, 5132 may be any voltagevalues indicative of the output value for that parameter having theappropriate range and scaling to provide the functions described herein.

Regarding the location and movement of the DNA 4810 at each of the fivetime states T1-T5 in the example of FIG. 51, at time stage T1 (5102)=noDNA in nanopore (Open Pore), at time stage T2 (5104)=Base A in nanopore,at time stage T3 (5106)=Base G in nanopore, at time stage T4 (5108)=BaseA in nanopore, and at time stage T5 (5110)=No DNA in nanopore (OpenPore).

Referring to FIGS. 50 and 51, starting at time stage T1, the DNA 4810 isoutside of the pore 4808, the capacitance to ground is low (as discussedherein), the resonance frequency f high, and the measured magnitudeoutput signal is high and the phase value is very low (e.g., about isabout 0 deg.) at the monitoring frequency f_(probe), which correspondsto the response curves 5018, 5028 shown in FIG. 50, having output valuesat f_(probe) where the curves 5018, 5028 intersect with the line 5004,corresponding to an output voltage V1 and output phase Ph5,respectively. At time T2, the DNA base 4816 (Base A, in this example)enters the pore 4808, the capacitance to ground increases, the resonancefrequency f_(res) shifts to lower central frequency, and the measuredmagnitude output signal is low and the phase value is intermediate(e.g., about 90 deg.) at f_(probe), which corresponds to the responsecurves 5012, 5022 shown in FIG. 50, having output values at f_(probe)where the curves 5012, 5022 intersect with the line 5004, correspondingto an output voltage V5 and output phase Ph2, respectively. At time T3,the DNA base 4814 (Base G, the largest base, in this example), entersthe pore 4808, the capacitance to ground extremely high, and theresonance frequency f_(res) shifts lower, but the magnitude outputsignal is an intermediate output value is upper-mid (e.g., about 135deg.) at f_(probe), which corresponds to the response curves 5010, 5020shown in FIG. 50, having output values at f_(probe) where the curves5010, 5020 intersect with the line 5004, corresponding to an outputvoltage V2 and output phase Ph1, respectively.

At time T4, the DNA base 4812 (Base A in this example) enters the pore4808 (again), the capacitance to ground decreases (from the value atT3), the resonance frequency f_(res) shifts to central frequency, andthe measured magnitude output signal is low and the phase value isintermediate (e.g., about 90 deg.) at f_(probe), which corresponds tothe response curves 5012, 5022 shown in FIG. 50, having output values atf_(probe) where the curves 5012, 5022 intersect with the line 5004,corresponding to an output voltage V5 and output phase Ph2,respectively. At time T5, the DNA 4810 is outside of the pore 4808(again), the capacitance to ground is low, the resonance frequencyf_(res) shifts (again) to high frequency, and the measured magnitudeoutput signal is high at f_(probe), which corresponds to the responsecurves 5018, 5028 shown in FIG. 50, having output values at f_(probe)where the curves 5018, 5028 intersect with the line 5004, correspondingto an output voltage V1 and output phase Ph5, respectively.

Referring to FIG. 52, another overview example 5200 of a time sequence(T1-T5) is shown for reading data stored in DNA using two-bits (e.g.,two bases) in the DNA strand via the capacitive-resonance DNA datareading technique of the present disclosure using a different fixedprobe (or monitor) frequency f_(probe2). The sequence has 5 timesegments or stages T1-T5, shown as five columns 5202-5210, and theprogression between time segments is shown by dashed lines 5211. Foreach of the time stages T1-T5, there is an image showing the DNA 4810(FIG. 48) and its location relative to the nanopore 4808 for that timestage, there are also magnitude and phase response curves 5212-5220 forthe time stages T1-T5, respectively, showing the intersection of theresponse curve at the monitor frequency f_(probe2) (corresponding to acurve from the family of curves shown in FIG. 50), and two value graphs5230, 5232 showing corresponding output signals (e.g., voltage values)for magnitude and phase responses, respectively, at the monitorfrequency f_(probe2) across each of the time stages T1-T5. The valuesV1-V5 and Ph1-PH5 for the graphs 5130, 5132 may be any voltage valuesindicative of the output value for that parameter having the appropriaterange and scaling to provide the functions described herein.

Regarding the location and movement of the DNA 4810 at each of the fivetime states T1-T5 in the example of FIG. 52, more specifically, at timestage T1 (5202)=no DNA in nanopore (Open Pore), at time stage T2(5204)=Base A in nanopore, at time stage T3 (5206)=Base G in nanopore,at time stage T4 (5208)=Base A in nanopore, and at time stage T5(5210)=No DNA in nanopore (Open Pore).

Referring to FIGS. 50 and 52, starting at time stage T1, the DNA 4810 isoutside of the pore 4808, the capacitance to ground is low (as discussedherein), the resonance frequency f_(res) is high, and the measuredmagnitude output signal is high and the phase shift value is very low(e.g., about 0 deg.), which corresponds to the response curves 5018,5028 shown in FIG. 50, having output values at f_(probe2) where thecurves 5018, 5028 intersect with the line 5006, corresponding to anoutput voltage V6 and output phase Ph10, respectively. At time T2, theDNA base 4816 (Base A, in this example) enters the pore 4808, thecapacitance to ground increases, the resonance frequency f_(res) shiftsto lower central frequency, and the measured magnitude output signal isintermediate and the phase value is low-mid value (e.g., about 45 deg)at f_(probe2), which corresponds to the response curves 5012, 5022 shownin FIG. 50, having output values at f_(probe2) where the curves 5012,5022 intersect with the line 5006, corresponding to an output voltage V9and output phase Ph7, respectively.

At time T3, the DNA base 4814 (Base G, the largest base, in thisexample), enters the pore 4808, the capacitance to ground is extremelyhigh, and the resonance frequency f_(res) shifts lower, and themagnitude output signal at f_(probe2) is at a low output value and theoutput phase is at about an intermediate or mid value (e.g., about 90deg.), which corresponds to the response curves 5010, 5020 shown in FIG.50, having output values at f_(probe2) where the curves 5010, 5020intersect with the line 5006, corresponding to an output voltage V10 andoutput phase Ph6, respectively. At time T4, the DNA base 4812 (Base A,in this example) enters the pore 4808 (again), the capacitance to grounddecreases (from value at T3), the resonance frequency f_(res) shifts tocentral frequency, and the measured magnitude output signal isintermediate and the phase is low at f_(probe2), which corresponds tothe response curves 5012, 5022 shown in FIG. 50, having output values atf_(probe2) where the curves 5012, 5022 intersect with the line 5006,corresponding to an output voltage V9 and output phase Ph7,respectively. At time T5, the DNA 4810 is outside of the pore 4808, thecapacitance to ground is low, the resonance frequency f_(res) shifts toa higher frequency, and the measured magnitude output signal is high andthe phase signal is very low (e.g., about 0 deg.) at f_(probe2), whichcorresponds to the response curves 5018, 5028 shown in FIG. 50, havingoutput values at f_(probe2) where the curves 5018, 5028 intersect withthe line 5006, corresponding to an output voltage V6 and output phasePh10, respectively.

Referring to the two examples of FIGS. 51 and 52, and comparing theoutput graphs 5130,5132, and 5230,5232, it can be seen that the desiredoutput values can be selected based on the measurement or monitoringfrequency f_(probe), f_(probe2).

While DNA using two and four bits (or bases) representing data to beread have been described above, any number of “bits” (or monomers orbases) may be used if desired for the data storage polymer, provided thechange in cell capacitance or impedance (and corresponding resonancefrequency, or frequency response) is sufficient to produce an outputmagnitude and/or phase for each bit that is distinguishable over each ofthe other bits. While such capacitance change may be accomplished bychanging the physical molecular size of the bases (e.g., the diameter),any property of the bases that creates a unique capacitance value of thecell when passing through the nanopore may be used if desired. Forexample, bases that have different dielectric properties, differentionic (or charge) properties, and/or different quantummechanical/electrical properties, may be used, provided they meet thedesired functional and performance requirements.

Referring to FIG. 53, an equivalent circuit and block diagram 5300 of aDNA data reading network array of the present disclosure is shown,having a parallel array of resonant circuits or Nanopore PolymerResonators (NPRs) 5302-5306 (NPR1-NPR3) each connected in parallel,through a individual coupling capacitors C_(CPL) (discussedhereinafter), to a common AC input voltage source 5308, which providesan AC voltage Vin having a frequency that includes at least the desiredmeasurement frequency(ies) (f_(probe)) for each of the resonant circuitsNPR1-NPR3 (discussed more hereinafter). Each of the resonators NPR1-NPR3has a unique inductor values L1-L3, respectively, connected in serieswith equivalent circuits 5312-5316 of corresponding cells (similar tothe cell 4800 described in FIGS. 48A-48C, and similar to the equivalentcircuit 4900 described in FIG. 49A), having a variable capacitor C andvariable resistor R, which vary based on the location of the polymer (orDNA) 4810 (FIG. 48A) in the cell 4800 with respect to the nanopore 4808in the cell 4800 (as discussed herein above). Each of the uniqueinductor values L1-L3 sets a unique resonant frequency band Δf_(res)(FIG. 50) and a corresponding overall resonator bandwidth Δf_(BW) (FIG.50), discussed more hereinafter with FIG. 54. Thus, the plurality ofresonators NPR1-NPR3 are connected in parallel in afrequency-multiplexed (or frequency-division multiplexed) arrangementcreating an array of the resonators NPR1-NPR3, all driven by a single ACinput voltage 5308, and each responding to its own probe inputfrequency.

Referring to FIG. 53, an optional AC RF attenuator 5310 may be providedin series with the AC input source 5308 prior to the connection to theparallel array of resonators NPR1-NPR3 to provide a voltage divider orimpedance matching with the resonators NPR1-NPR3 and/or to adjust the ACoutput voltage Vout range based on the range of impedance values of theresonators over the operational frequency range of interest. Theattenuator 5310 may be a constant or switched or variable type RFattenuator, depending on the frequency range used, the impedance of thearray and load, and/or the desired functional and performancecharacteristics.

Referring to FIG. 53, an AC output voltage Vout from the parallel arrayof resonators NPR1-NPR3, may be provided to an amplifier (or pre-amp)5320, which performs signal conditioning on the output signal Vout, suchas remove noise, filter around the measurement frequency(ies) ofinterest, isolate impedance of resonator array from down-stream devicesor components, improve measurement sensitivity, amplify or attenuate theVout signal, and/or perform other desired signal conditioning of the ACoutput voltage signal Vout as needed to provide the desired functionsand/or performance. In some embodiments, the amplifier may also be anactive filter which filters the AC output voltage signal around one ormore of the probe frequencies. The amplifier 5320 provides an analog ACconditioned output voltage Vout signal on line 5322 to an A/D Converter5324 (e.g., an integrated circuit or chip), which digitally samples theconditioned AC output voltage Vout and provides digital output data on aline 5326 indicative of the sampled conditioned AC output voltage Voutsignals. The sample rate of the A/D converter may be any rate thatprovides sufficient sampling of the output signal to preserve theability to perform frequency analysis at the desired measurementfrequencies (e.g., the probe frequencies). The AC output voltage mayalso be down-converted to a lower intermediate frequency or DC, e.g., ifthe fundamental probe frequency(ies) is/are too high (or fast) to bedirectly sampled accurately by the A/D converter (or for other design orperformance reasons), by mixing the AC output signal with the same (orsimilar) frequency, e.g., homodyne or heterodyne demodulation, or anyother type or demodulation or frequency conversion, provided itpreserves the magnitude and/or phase components needed to accuratelymeasure the desired parameters. The A/D converter 5324 may have on-boardmemory that stores the sampled output data and/or may be connected to orcommunicate with a separate memory device (not shown) which may storeall or a portion of the sampled output data. The digital sampled outputdata is provided on the line 5326 to digital signal processing frequencyanalysis (or decomposition) logic 5328, such as an FFT (or Fast FourierTransform) logic or chip, which performs digital signal processing (DSP)on the digital sampled data and provides digital data on a line 5330indicative of the magnitude and/or phase of the frequency components (orharmonics) that exist in the sampled AC output signal Vout. Instead ofthe FFT logic 5328, any other frequency analysis hardware, firmwareand/or software may be used if desired, provided it provides thefunctions and performance described herein and adequately measures themagnitude and/or phase of the output signal at the desired frequenciesof interest (e.g., at least at the desired probe or measurementfrequencies).

The amplifier 5320, the A/D converter 5324, and the FFT logic 5329 areall known hardware or firmware components (which may have computerprogrammable portions) that may be obtained from an integrated circuitprovider, such as Texas Instruments, Inc., Analog Devices, Inc.,National Instruments Corp., Intel Corp, or other similar manufacturers.One example of components for digitization that may be used include:Xilinx FFT LogiCORE, part no. 4DSP FMC103, 1126, Alazartec 9360, 9370.Other components may be used if desired, provided they provide thefunctions and performance described herein. Also, the FFT logic may beperformed by a field programmable gate array (FPGA).

Also, instead of sampling the output voltage with an A/D converter andperforming digital signal processing to determine the frequencycomponents, the output signal Vout may be provided to one or more analogfilters (not shown) tuned to the desired frequencies to identify themagnitude and/or phase of the desired frequency components and providean analog output voltage signal indicative of same.

Referring to FIG. 54, a frequency plot 5400 shows a sample frequencyseparation for the plurality of resonators NPR1-NPR3 of FIG. 53 of thepresent disclosure. In particular, as discussed above with FIG. 53, eachof the resonators NPR1-NPR3 has a unique resonator bandwidth Δf_(BW1),Δf_(BW2), Δf_(BW3), which are set or determined by the unique inductorvalues L1-L3, respectively. The bandwidths Δf_(BW1), Δf_(BW2), Δf_(BW3),of the resonators NPR1-NPR3, respectively, may be separated fromadjacent resonator bandwidths by a frequency separation or gap Δf_(gap)such that the resonator bandwidth Δf_(BW) of adjacent resonators do notoverlap and cause interference or cross-talk between the adjacentresonators. In some embodiments, the bandwidths may overlap provided thefrequency response of each base is different and thus, they do notaffect the ability to identify the response of each resonator.

Similarly, in FIG. 54 there is shown a set of probe or monitorfrequencies f_(p1),f_(p2),f_(p3) corresponding to the bandwidthsΔf_(BW1), Δf_(BW2), Δf_(BW3), of the resonators NPR1-NPR3, respectively,which may be a frequency (or frequencies) within each of the resonatorbandwidths Δf_(BW1), Δf_(BW2), Δf_(BW3), that provides the desiredoutput signals as discussed herein, such as in the range of resonantfrequencies seen when the polymer is in the nanopore (as discussedherein above with FIGS. 50-52). Other measurement or probe frequenciesmay be used if desired provided it meets the desired functional andperformance requirements.

While the gap frequency is determined primarily by the choice ofinductor L value, the amount of separation between adjacent frequencybands needed to avoid undesired overlap during operation caused bysystem parameter variations that may occur over the system operatingconditions may also be determined by various factors, including but notlimited to cell design parameter tolerances (e.g., electrodes,inductors, capacitors, fluids, cell walls, membranes, materials,dimensions, and any other cell design parameters that may vary from cellto cell), environmental operating ranges, e.g., temperature, pressure,humidity, and the like, electro-magnetic interference or noiseparameters/effects, any cell-to-cell (or chamber-to-chamber)interactions, and/or any other design practice, safety or regulatoryrequirements or tolerances, or any other factors that may affect thedesired function or performance. Such factors can cause the bandwidth ofa given resonator to change from its ideal conditions, and thus shouldbe considered in the overall design tolerance parameters for thefrequency separation over time and environments.

Referring to FIGS. 55A and 55B, the AC input voltage Vin of the presentdisclosure includes at least desired measurement or probe frequencies atwhich the output AC voltage Vout will be frequency analyzed (e.g., bythe FFT logic 5330—FIG. 53). Referring to FIG. 55A, the AC input voltageVin may be a continuous broadband AC frequency signal shown by a curve5502 having all frequencies from the minimum possible measurementfrequency f_(MIN) to the minimum possible measurement frequency f_(MAX),or from the first probe frequency f_(p1) to the last probe frequencyf_(pN). In that case, all the resonators NPR1-NPR3 are excited by thebroadband frequency signal and will exhibit a response in the frequencycomponents of the output signal. Alternatively, the AC input voltage Vinmay be a broadband AC frequency signal shown by a curve 5510 having onlythe desired probe or monitor frequencies f_(p1),f_(p2),f_(p3),f_(pN) fas shown by the individual frequency components 5512,5514,5516, 5518,respectively. Also, the overall frequency range for the AC input voltageVin for all the resonators in the array may be from about 1.0 MHz to 1GHz. Other frequencies may be used if desired, provided it meets thefunction and performance described herein. The AC input voltage Vin maybe provided by a known oscillator chip (which may be adjustable and/orprogrammable) that provides the desired AC frequency components for thedesired design configuration and excitation, such as FMC 2850, TIDAC900,or Xilinx DS558. For the case where Vin contains a plurality ofindividual probe frequencies, Vin may be created by combining separateAC frequencies together electronically, or directly synthesizedmathematically and programmed into the oscillator (either hard wired orprogrammed by a microprocessor connected thereto).

Referring to FIG. 55B, the AC input voltage Vin may be a time swept ACfrequency signal shown by a curve 5550, sweeping the input frequencyfrom the minimum possible measurement frequency N_(MIN) to the minimumpossible measurement frequency f_(MAX), or from the first probefrequency f_(p1) to the last probe frequency f_(pN), and then repeatedhaving a repeat-period of time T. In that case, the frequency of theinput voltage Vin is at only one frequency at any given time, and allthe resonators NPR1-NPR3 respond to that single input frequency and willexhibit a response to that frequency at the output signal. Also, in thatcase, because the system only responds to one frequency at a time, andthe system knows the frequency sweep timing of the input voltage Vin,there is no need for frequency analysis as the system can sample themagnitude and/or phase at the time associated with the desired probefrequency and determine the value directly.

Alternatively, the AC input voltage Vin may be a time-stepped ACfrequency signal shown by a curve 5570, where the input frequency isstepped from the first probe frequency f_(p1) to the last probefrequency f_(pN), and waits a predetermined dwell time T_(D) at eachfrequency, and then is repeated having a repeat-period of time T. Inthat case, similar to the swept-frequency curve 5550, the frequency ofthe input voltage Vin is at only one frequency at any given time, allthe resonators NPR1-NPR3 respond to that single input frequency and willexhibit a response to that frequency at the output signal at that time.The dwell time T_(D) allows more time for system to sample the outputsignal at each probe frequency. Also, in that case, because the systemonly responds to one frequency at a time, and the system knows thetiming, there is no need for frequency analysis (or decomposition) asthe system can sample the magnitude and/or phase at the time associatedwith the desired probe frequency and determine the value directly.

Also, the overall frequency range for the AC input voltage Vin for allthe resonators (and also the probe measurement frequency) in the arraymay be from about 1.0 MHz to 1 GHz. Other frequencies may be used ifdesired, provided they meet the functional and performance requirementsdescribed herein. The AC input frequency should be set at a value thatenables sufficient number of cycles (or periods) of the input frequencyto allow the impedance of the cell to be adequately sampled. This willbe based in part on the speed at which the DNA (or other polymer) ismoving through the nanopore. For example, if the DNA is moving throughthe nanopore at a rate of about 1 MHz (i.e., one million bases everysecond), and if the AC input frequency is 100 MHz, then the cellimpedance will receive (or experience) 100 cycles of the input frequencyfor each base, which corresponds to a 100:1 “sample” rate. Other inputfrequencies and sample rates may be used if desired, provided theyprovide the desired function and performance. For example, the minimumsample frequency required to digitally resolve a given input frequencyis the Nyquist sample frequency, which is 2× the input frequency. Inthis case, for a 1 MHz input signal (rate of DNA passage throughnanopore), the minimum (or Nyquist) sample rate would be 2 MHz.

Referring to FIG. 56, an example of frequency spectrum graphs of the ACoutput voltage Vout magnitude and phase 5600, 5620, respectively, areshown, for some embodiments of the present disclosure. In particular,for the AC output voltage signal Vout, there are three magnitudes lines5602,5604,5606 shown, corresponding to the frequency responses of NPR1,NPR2, NPR3, respectively. For this example, the probe frequency used isf_(probe2), and there are only two bits, like the example shown in FIG.52. At the time this output was obtained for this example, NPR1 had thescenario shown in column 5206 at time T3 (FIG. 52), and thecorresponding frequency response line 5602 indicates a magnitude of V10and a phase of Ph6. At the same time, NPR2 had the scenario shown incolumn 5204 at time T2 (FIG. 52), and the corresponding frequencyresponse line 5604 indicates a magnitude of V9 and a phase of Ph7. Alsoat the same time, NPR3 had the scenario shown in column 5210 at time T5(FIG. 52), and the corresponding frequency response line 5606 (FIG. 56)indicates a magnitude of V6 and a phase of Ph10.

In some embodiments, the probe or measurement frequency used may varybased on the resonator frequency response, polymer properties, and otherfactors such as system noise that may affect the quality of the outputsignal. In some embodiments, the system may switch between measurementfrequencies in real time to ensure the best quality output signal isobtained, or multiple different measurement frequencies may be used toperform error checking or validation of the data read. In that case, theAC input frequency of Vin should include the measurement frequency,e.g., by changing or adjusting accordingly (in synch with themeasurement) or having the measurement frequency be part of thecontinuous AC input frequency components provided.

Referring to FIG. 57, a top level block diagram 5700 is shown forembodiments of the present disclosure. In particular, when the array ofresonators (or NPRs) is laid-out on a chip, it may be configured in a2-Dimensional array of M×N resonators, where there are M rows 5702-5708and each row having N resonators, all connected in parallel (as shown bylines 5716) and all NPRs driven by the same AC input voltage Vin 5308(FIG. 53) on the line 5710 and all NPRs contributing to a commonfrequency division multiplexed AC output voltage Vout on a line 5712,which may be fed to the amplifier (or pre-amp) 5320 (FIG. 53). In thatcase, each of the rows 5702-5708 may correspond to a frequency band,such as 100 MHz-199 Mhz (for row 5702), 200 MHz-299 MHz (for row 5704),300 Mz-399 MHz (for row 5706), and the like for the other rows. Withineach of the rows 5702-5708, there may be a plurality of resonators (orNPRs), each shown as a box 5714 with the designation f_(row,column).Each of the NPRs in a given row have a resonator bandwidth Δf_(BW)within the frequency band associated with that row, and is separated infrequency from the adjacent NPR by a gap frequency band Δf_(gap), toavoid interference or cross-talk between the adjacent resonators, asdiscussed herein above with FIG. 54.

For example, if the first row at the top 5702 is designated with thefrequency band 100 MHz-199 Mhz, the NPRs f_(1,1) to f_(1,N) in that row5702 would be within this band and be separated from each other by a gapfrequency band, as discussed herein above and with FIG. 54. Thus, inthis format, the reading system may be viewed as a 2D array of dataelements that are individually readable with a single input and outputline. Other frequency bands and ranges may be used if desired.

Referring to FIG. 58, a cross-sectional view of a multi-layer chipstructure 5800 is shown, including the cell 4800 (FIG. 48A), theinductor L (FIGS. 49A, 53), and the coupling capacitor C_(CPL), andincluding a top contact 5802, where the input (and output) I/O voltageline may be connected, which may collectively referred to herein as thenanopore-polymer resonator (NPR). Matching components of the cell 4800(FIG. 48A) are labeled the same in FIG. 58. Above the upper electrode4818 is a vertical connection 5806 to the center of the chip inductor5808 (see FIG. 59) L and the other end of the chip inductor 5808 isconnected to coupling chip capacitor 5812. The upper side of the chipcapacitor 5812 is connected to the I/O contact 5802. 3-D stacking ofmultiple layers allows for increased packing of the cell 4800 andcircuit components which permits close packing of the cells 5804. Inparticular, referring to FIGS. 48A and 58, the cell 4800. There may alsobe dielectric layers 5804 that separate each of the functional circuitelements. A plurality of copies of the resonator or NPR structure 5800may be connected together, in one or two dimensional arrays, to create a“chip” having the NPR arrays discussed herein above, e.g., with FIGS. 53and 57. Also, the amplifier 5320 (FIG. 53), e.g., a CMOS amp or pre-amp,may be integrated into the chip structure in the output line contactlayer 5802 at an appropriate location, e.g., after the last NPR 5800 inthe array, via “flip chip” bonding or any other technique that providesthe desired function and performance. Also, Referring to FIG. 59, a topview of the inductor L that may be used in the chip 5800 is shown, whichmay be fabricated using known chip-inductor fabrication techniques, suchas lithographic fabrication or other fabrication techniques. Asdiscussed herein, there may also be a DC voltage applied to theelectrodes to move or steer the DNA or polymer floating in the chambersto a particular desired chamber. As discussed herein, the AC voltage isapplied to all the NPRs 5800 via the I/O contact which may be common toall NPRs and which is fed by the AC input voltage on a line 5812, andthe DC voltage may be applied individually to each electrode on a line5810, with each NPR have its own separate DC voltage input line 5810 touniquely control the electrode 4818.

Referring to FIGS. 60 and 61, to allow both the AC and DC voltages todrive the same cell, the AC and DC lines may be connected to thestructure 5800 using a “bias tee” connection, having a circuit shown inFIG. 60 and a sample physical chip implementation shown in FIG. 61.Referring to FIG. 60, the AC RF (high frequency) input signal Vin iscoupled to the inductor L through a coupling capacitor C_(CPL) (asdiscussed previously herein) and the DC input may be connected to thesame side of the inductor L through a highly resistive wire Rw, whichhas enough self-inductance to “block” the high frequency AC signals fromleaving the circuit via the DC input source path. Alternatively, theresistive wire Rw may be connected to the other side (electrode side) ofthe inductor L, if desired. However, in that case, the value of theresistive wire Rw would act to dampen the resonance (as another resistorin parallel with the cell capacitance to AC ground).

Referring to FIG. 61, an example of a physical implementation of the“bias tee” connection is shown, which shows a magnified view of the“bias tee” connection. High frequency AC input signal Vin iscapacitively coupled to the inductor L via a transmission line (e.g.,the top I/O contact 5802—FIG. 58) with a gap to a second plate 6102 tocreate the coupling capacitor CCPL which coupled the AC and blocks theDC voltage. Also, the DC input voltage (or DC “steering” voltage) may beconnected to the same side of the inductor L through the highlyresistive wire Rw, which has enough self-inductance to “block” the highfrequency AC signals from leaving the circuit via the DC input sourcepath, as also discussed above with FIG. 60. The result of the “bias tee”connection is that the voltage applied to the inductor L is AC inputvoltage having a DC bias determined by the DC input voltage.

Referring to FIG. 62, a cross-sectional view of a multi-layer chipstructure 6200 is shown, including a cell having three chambers similarto that shown in FIGS. 24, 25, 28, and 29 and described herein, andhaving two integrated inductors L1A,L1B. In that case, there is an upper(or top) left chamber 6202 with a top left electrode 6210 (for adding abit, e.g., “0”), a top right chamber 6204 with a top right electrode6212 (for adding a bit, e.g., “1”), and a lower “de-blocking” chamber6206, common to both top left 6002 and top right 6004 chambers, with acorresponding electrode 6214, which may be connected to ground (e.g., 0volts). For the three-chamber cell, it can be viewed as having twocapacitors in parallel, each having their own impedance which changeswith time. In this case, the left inductor L1A is connected to the lefttop electrode 6210, and the right inductor L1B is connected to the righttop electrode 6212. The remainder of the components and elements may bethe same as discussed hereinbefore with the two-chamber cell design. TheAC RF I/O input line may be capacitively coupled to the inductor witheach DC input line coupled by a resistor or resistive wire, using the“bias tee” connection as discussed herein above. If the inductors L1A,L1B have different values, the left and right chambers will havedifferent resonant frequencies and different resonant bandwidths. Inthat case, each three-chamber cell would have two resonators with tworesonant bandwidths, that may be positioned in the frequency space to beinterrogated or monitored to read the data on the polymer (or DNA) asdiscussed herein above. If the inductors L1A, L1B have the same values,the DNA can still be read for each chamber because the system can onlyread one chamber at a time, as there is only one polymer (or DNA) strandper cell. So it is inherently time sequenced or time dependent, and thusthey do not need to be separated in frequency to accomplish the datareading. However, it may be desirable to separate them to ensure (orvalidate) the correct chamber is actually being read.

Referring to FIG. 63, a cross-sectional view of a multi-layer chipstructure 6300 is shown, including a three-chamber cell having similarto that shown in FIG. 62, having a single integrated inductor L1A. Insome embodiments, depending on the data read and write protocol, it maybe desirable to use only one cell, e.g., the top left cell 6002, to readthe polymer data, and the other cell, e.g., 6004, may be not beconfigured for reading, and thus does not have an inductor and does notform a resonator. In that case, the AC input voltage Vin and the DCsteering voltage, may be coupled to the inductor L1A using the “biastee” connection, as described herein above with other embodiments, todrive the left top electrode 6210, the DC steering voltage coming in online 6302, and for the right top electrode 6212, the DC steering inputvoltage may be connected directly as shown by a line 6304. The rest ofthe components and elements may be the same as discussed hereinbeforewith FIG. 62 for the three-chamber cell design.

Referring to FIGS. 64 and 64A, instead of having the inductor attachedto one or two of the top electrodes, a single integrated inductor L1 maybe connected to the bottom electrode 6214 and the top electrodes6210,6212 are connected individually to the respective DC steeringvoltage for that electrode on lines 6410,6412, respectively. In thatcase, there would be “bias tee”—type connection at the top and bottom ofthe circuit (see FIG. 64A). In that case, the AC RF input voltage may beprovided to the bottom electrode, which may be AC coupled to theinductor L1 via a coupling capacitor C_(CPL) and a coupling capacitor atthe top couples the AC to AC RF ground. The DC lines 6410, 6412 (FIG.64), are each coupled through Rw (FIG. 64A) to their respectiveelectrode, and the DC is passed through the cell and inductor andthrough a bottom Rw to DC ground. The bottom contact may act as the ACrf I/O line, and the top contact 4610 may act as the DC I/O line. Also,the DC ground could also be a DC input line, the only requirement is todefine the DC potential difference between the top and bottomelectrodes. With a common inductor L1, the DNA can still be read foreach chamber because the system can only read one chamber at a time, asthere is only one polymer (or DNA) strand (or memory string, asdiscussed herein) per cell (as discussed above). Thus, it is inherentlytime sequenced or time dependent. Referring to FIGS. 63, 63A, and 63B,in some embodiments, instead of using a unique inductor L for eachresonator (such as is shown in FIG. 63) to set unique resonancefrequency and frequency bandwidth, a single common inductor L_(common)may be used for all resonators in the array and a unique capacitor C_(R)may be provided in parallel to each cell having a value that sets theresonant frequency of each resonator. In that case, the memory chip mayhave a built-in fixed chip resonator capacitor C_(R) from the topelectrode to the bottom electrode for each chamber being measured to setthe resonance frequency with the common inductor for each cell. As thepolymer moves through the nanopore, and the capacitance of the cellchanges, this capacitance change would adjust the overall parallelcapacitance combination and adjust the resonance frequency accordingly.Like the case with the unique inductors L, the value of the fixedresonance capacitor C_(R) would be set to provide a unique resonancefrequency response for each resonator in the resonator array. Asdiscussed for the inductor embodiment herein above, there may only needto be one capacitor (one resonator) that performs the measurement, or iftwo are used, they may be the same value (due to inherent timesequencing as discussed above), or may be different values if desiredfor validation, redundancy or other purposes. FIG. 63A shows an exampleof an equivalent circuit diagram for several cells with fixed resonancecapacitors C_(R1), C_(R2),C_(R3), and a common inductor L_(common).

Referring to FIGS. 64, 64B, 64C, in some embodiments, instead of using aunique inductor L on the bottom of each resonator (such as is shown inFIG. 64) to set unique resonance frequency and frequency bandwidth, asingle common bottom inductor L_(common) may be used for all resonatorsin the array and a unique capacitor C_(R) may be provided in parallel toeach cell having a value that sets the resonant frequency of eachresonator. a similar variation using a fixed may be done for theinductor at the bottom, where resonance capacitors C_(R1), C_(R2),C_(R3), and a common inductor L_(common) may be used. In that case, thememory chip may have a built-in fixed chip resonator capacitor C_(R)(FIG. 64C) from the top electrode to the bottom electrode for eachchamber being measured to set the resonance frequency with the commoninductor L_(common) for each cell.

The present disclosure does not require the cells to be individuallyaddressable to read the data in each of the cells. Also, the presentdisclosure allows the reading of data stored on polymers located in eachof the cells by using a single source input line and single output line,using frequency division multiplexing. Further, the data readingtechnique of the present invention will work with any type of nanopore,e.g., solid state, protein-based, or any other type of nanopore. Inaddition, the system and method of the present disclosure uses high rffrequencies to read the memory string (or DNA or polymer), e.g., about 1MH-1 GHz, which substantially eliminates the 1/f noise, so the systemwill likely have higher sensitivity (or granularity or fidelity) thansystems not using such a high frequency measurement approach. Inaddition, using such a high frequency approach also provides a fast timescale for reading (or sampling) the memory string as it passes throughthe nanopore, thereby not requiring the string to be intentionallyslowed down for sampling or measurement purposes.

In some embodiments, the invention provides a nanochip for sequencing anelectrically charged polymer, e.g., DNA, comprising at least twodistinct monomers, the nanochip comprising at least a first and secondreaction chambers, each comprising electrolytic medium, and separated bya membrane comprising one or more nanopores, wherein a pair ofelectrodes (for example in the form of opposing plates), connected incircuit, is disposed on either side of the membrane comprising one ormore nanopores, the electrodes being separated by a distance of 1-30microns, e.g., about 10 microns, such that the gap between theelectrodes has a capacitance when a radiofrequency pulsating directcurrent, e.g. 1 MHz to 1 GHz, is applied to the electrodes so as to drawthe electrically charged polymer through the nanopore, e.g., from onechamber to the next, and such that the phase of the pulsating directradiofrequency current changes with changes in capacitance as theelectrically charged polymer passes through the nanopore, therebyallowing detection of the monomer sequence of the electrically chargedpolymer. In certain embodiments, the nanochip comprises multiple sets ofreaction chambers wherein the reaction chambers within a set areseparated by membrane having one or more nanopores, and the sets ofreaction chambers are separated by a screening layer to minimizeelectrical interference between the sets of reaction chambers and/or toseparate multiple linear polymers and allow them to be sequenced inparallel.

For example, in one embodiment the electrodes form the top and bottomplates of a capacitor embedded in a resonant circuit, and the change incapacitance is measured as the DNA passes through the pore between theplates.

In certain embodiments, the nanochip further comprises reagents forsynthesizing the polymer, e.g. DNA, e.g., according to any of Nanochip1, et seq., below.

In one embodiment, therefore the invention provides a method (Method 1)for synthesizing a charged polymer [e.g., a nucleic acid (e.g., DNA orRNA)] comprising at least two distinct monomers in a nanochip, e.g., ananopore-based device, e.g., any of Nanochip 1, et seq., the nanochipcomprising

-   -   one or more addition chambers containing reagents for addition        of one or more monomers [e.g. nucleotides] or oligomers [e.g.,        oligonucleotides] to the charged polymer in a buffer solution in        terminal protected form, such that only a single monomer or        oligomer can be added in one reaction cycle; and    -   one or more reserve chambers containing buffer solution but not        all reagents necessary for addition of the one or more monomers        or oligomers,    -   wherein the chambers are separated by one or more membranes        comprising one or more nanopores and    -   wherein the charged polymer can pass through the nanopore but        the least one of the reagents for addition of one or more        monomers or oligomers cannot,    -   the method comprising    -   a) moving the first end of a charged polymer having a first end        and a second end, by electrical attraction, into an addition        chamber, whereby monomers or oligomers are added to said first        end in blocked form,    -   b) moving the first end of the charged polymer with the added        monomer or oligomer in blocked form into a reserve chamber,    -   c) deblocking the added monomer or oligomer, and    -   d) repeating steps a-c, wherein the monomers or oligomers added        in step a) are the same or different, until the desired polymer        sequence is obtained.

For example, the invention provides

-   -   1.1. Method 1, wherein the polymer is nucleic acid, e.g.,        wherein the polymer is DNA or RNA, e.g., wherein it is DNA, e.g        dsDNA or ssDNA.    -   1.2. Any foregoing method wherein the second end of the polymer,        e.g. the nucleic acid, is either protected or bound to a        substrate adjacent to the nanopore.    -   1.3. Any foregoing method wherein the electrical attraction is        provided by applying an electric potential between the        electrodes in each chamber, wherein the polarity and current        flow between the electrodes can be controlled, e.g., such that        the nucleic acid is attracted to a positive electrode.    -   1.4. Any foregoing method wherein the polymer is a nucleic acid        and        -   (i) the said first end of the nucleic acid is the 3′-end,            the addition of nucleotides is in the 5′ to 3′ direction and            is catalyzed by a polymerase, e.g., wherein the polymerase            is hindered (e.g. due to its size or due to being tethered            to a substrate in the first chamber) from passing through            the nanopore, the nucleotides are 3′-protected when added,            and following addition of the 3′-protected nucleotide to the            3′-end of the nucleic acid, the 3′-protecting group on the            nucleic acid is removed, e.g., in the reserve chamber; or        -   (ii) the said first end of the nucleic acid is the 5′ end,            the addition of nucleotides is in the 3′ to 5′ direction,            the nucleotides are 5′-protected when added, and following            addition of the 5′-protected nucleotide to the 5′-end of the            nucleic acid, the 5′protecting group is removed, e.g., in            the second chamber; (for example wherein the phosphate on            the 5′-protected nucleotide is a nucleoside phosphoramidite            coupled via the 5′-protecting group to a bulky group which            cannot pass through the nanopore, so that following coupling            to the nucleic acid, the unreacted nucleotides are flushed            away, the bulky 5′-protecting group is cleaved from the            nucleic acid, and flushed away, and the 5′-end of the            nucleic acid can be moved into the reserve chamber);        -   wherein the addition of nucleotides to the nucleic acid is            controlled by movement of the first end of the nucleic acid            into and out of the one or more addition chambers, and the            cycle is continued until the desired sequence is obtained.    -   1.5. Any foregoing method wherein the sequence of monomers or        oligomers in the polymer [e.g., the sequence of nucleotides in        the nucleic acid] thus synthesized corresponds to a binary code.    -   1.6. Any foregoing method wherein the polymer thus synthesized        is single stranded DNA.    -   1.7. Any foregoing method wherein the sequence of the polymer        [e.g. the nucleic acid] is checked during the process or        synthesis by sequencing the monomers or oligomers [e.g.,        nucleotide bases] as they pass through the nanopore to identify        errors in sequencing.    -   1.8. Any foregoing method wherein the polymer thus synthesized        is single stranded DNA, wherein at least 95%, e.g at least 99%,        e.g., substantially all of the bases in the sequence are        selected from two bases that do not hybridize with other bases        in the strand, e.g. bases selected from adenine and cytosine.    -   1.9. Any foregoing method wherein a multiplicity of polymers        [e.g. oligonucleotides] are synthesized independently in        parallel, such that polymers [oligonucleotides] having different        sequences are obtained by separately controlling whether they        are present in one or more addition chambers or one or more        reserve chambers.    -   1.10. Any foregoing method wherein there are at least two        addition chambers that contain reagents suitable for adding        different monomers or oligomers, e.g. different nucleotides,        e.g., wherein there are one or more addition chambers containing        reagents suitable for adding a first monomer or oligomer and one        or more addition chambers containing reagents suitable for        adding a second different monomer or oligomer, for example        wherein there are one or more addition chambers containing        reagents suitable for adding adenine nucleotides and one or more        addition chambers containing reagents suitable for adding        cytosine nucleotides.    -   1.11. Any foregoing method wherein at least one addition chamber        is a flow chamber, providing a flow cycle comprising (i)        providing to the flow chamber reagents suitable for adding a        first monomer or oligomer, (ii) flushing, (iii) providing to the        flow chamber reagents suitable for adding a second different        monomer or oligomer, and (iv) flushing, and repeating the cycle,        until the synthesis is complete, wherein the sequence of        monomers or oligomers in the polymer is controlled by        introducing or excluding the first end of the polymer from the        flow chamber during step (i) or (iii) in each cycle;    -   1.12. Any foregoing method wherein the polymer is DNA and at        least one addition chamber is a flow chamber, providing a flow        cycle comprising (i) providing to the flow chamber reagents        suitable for adding a first type of nucleotide, (ii)        flushing, (iii) providing to the flow chamber reagents suitable        for adding a second type of nucleotide, and (iv) flushing, and        repeating the cycle until the synthesis is complete, wherein the        sequence is controlled by controlling the presence or absence of        the first end of the DNA (e.g. the 3′-end) in the flow chamber.    -   1.13. Any foregoing method wherein the polymer is DNA and at        least one addition chamber is a flow chamber, providing a flow        cycle comprising (i) providing to the flow chamber reagents        suitable for adding a first type of nucleotide, (ii)        flushing, (iii) providing to the flow chamber reagents suitable        for adding a second type of nucleotide, and (iv) flushing, (i)        providing to the flow chamber reagents suitable for adding a        third type of nucleotide, (ii) flushing, (iii) providing to the        flow chamber reagents suitable for adding a fourth type of        nucleotide, and (iv) flushing, and repeating the cycle until the        synthesis is complete, wherein the sequence is controlled by        controlling the presence or absence of the first end of the DNA        (e.g. the 3′-end) in the flow chamber when reagents suitable for        adding the different types of nucleotides are present.    -   1.14. Any foregoing method wherein the polymer is DNA and the        nanochip comprises two addition chambers which are flow        chambers, (a) the first flow chamber providing a flow cycle        comprising (i) providing to the first flow chamber reagents        suitable for adding a first type of nucleotide, (ii)        flushing, (iii) providing to the first flow chamber reagents        suitable for adding a second different type of nucleotide,        and (iv) flushing, and repeating the cycle until the synthesis        is complete, and (b) the second flow chamber providing a flow        cycle comprising (i) providing to the second flow chamber        reagents suitable for adding a third type of nucleotide, (ii)        flushing, (iii) providing to the second flow chamber reagents        suitable for adding a fourth different type of nucleotide,        and (iv) flushing, and repeating the cycle until the synthesis        is complete, wherein the nucleotides are selected from dATP,        dTTP, dCTP, and dGTP and wherein the sequence is controlled by        directing the first end of the DNA (e.g. the 3′-end) into the        flow chamber where the next desired nucleotide is provided.    -   1.15. Any foregoing method wherein the polymer is DNA and the        nanopore chip comprises one or more addition chambers for adding        dATP, one or more addition chambers for adding dTTP, one or more        addition chambers for adding dCTP, and one or more addition        chambers for adding dGTP.    -   1.16. Any foregoing method wherein the polymers [e.g. nucleic        acids] synthesized are each bound via their second end to a        surface proximate to a nanopore.    -   1.17. Any foregoing method wherein the sequence of the polymer        [e.g. nucleic acid] is determined following each cycle by        detecting the change in electric potential, current, resistance,        capacitance, and/or impedance as the polymer passes through the        nanopore.    -   1.18. Any foregoing method wherein the polymer is a nucleic acid        and synthesis of the nucleic acid takes place in a buffer        solution, e.g., a solution comprising a buffer for pH 7-8.5,        e.g. ca. pH 8, e,g, a buffer comprising        tris(hydroxymethyl)aminomethane (Tris), a suitable acid, and        optionally a chelator, e.g., ethylenediaminetetraacetic acid        (EDTA), for example TAE buffer containing a mixture of Tris        base, acetic acid and EDTA or TBE buffer comprising a mixture of        Tris base, boric acid and EDTA; for example a solution        comprising 10 mM Tris pH 8, 1 mM EDTA, 150 mM KCl, or for        example, 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM        Magnesium Acetate, pH 7.9 @ 25° C.    -   1.19. Any foregoing method wherein the polymer is single        stranded DNA further comprising converting the synthesized        single stranded DNA into double stranded DNA.    -   1.20. Any foregoing method further comprising removing the        polymer [e.g. the nucleic acid] from the nanochip after the        polymer synthesis is complete.    -   1.21. Any foregoing method wherein the polymer is a nucleic        acid, further comprising amplifying and retrieving copies of        synthesized nucleic acid using an appropriate primer and a        polymerase (e.g. Phi29).    -   1.22. Any foregoing method wherein the polymer is a nucleic        acid, further comprising cleaving the synthesized nucleic acid        with a restriction enzyme and removing the nucleic acid from the        nanochip.    -   1.23. Any foregoing method wherein the polymer is a nucleic        acid, further comprising amplifying the nucleic acid thus        synthesized.    -   1.24. Any foregoing method further comprising removing the        polymer [e.g., the nucleic acid] from the nanochip and        crystallizing the polymer.    -   1.25. Any foregoing method wherein the polymer is a nucleic        acid, further comprising stabilizing the nucleic acid, e.g., by        drying a solution comprising the nucleic acid together with one        or more of a buffer (e.g., a borate buffer), an antioxidant, a        humectant, e.g. a polyol, and optionally a chelator, for example        as described in U.S. Pat. No. 8,283,165 B2, incorporated herein        by reference; or by forming a matrix between the nucleic acid        and a polymer, such as poly(ethylene glycol)-poly(l-lysine)        (PEG-PLL) AB type block copolymer; or by addition of a        complementary nucleic acid strand or a protein that binds the        DNA.    -   1.26. Any foregoing method comprising:        -   (i) reacting a nucleic acid with a 3′-protected nucleotide            in an addition chamber, in the presence of a polymerase            which catalyzes the addition of the 3′-protected nucleotide            to the 3′ end of the nucleic acid;        -   (ii) drawing at least the 3′ end of the 3′-protected nucleic            acid thus obtained out of the addition chamber, through the            at least one nanopore, into a reserve chamber, wherein the            polymerase is hindered (e.g. due to its size or due to being            tethered to a substrate in the first chamber) from passing            through the nanopore;        -   (iii) deprotecting the 3′-protected nucleic acid, e.g.,            chemically or enzymatically; and        -   (iv) if it is desired that an additional 3′-protected dNTP            be added to the oligonucleotide, drawing the 3′ end of the            oligonucleotide into the same or different addition chamber,            so that steps (i)-(iii) are repeated, or if it is not so            desired, allowing the 3′ end of the nucleic acid to remain            in the reserve chamber until a further cycle wherein the            desired 3′-protected dNTP is provided to the addition            chamber; and        -   (v) repeating the cycle of steps (i)-(iv) until the desired            nucleic acid sequence is obtained.    -   1.27. Any foregoing method wherein the polymer is nucleic acid        single-stranded DNA (ssDNA) and the one or more nanopores have a        diameter allowing ssDNA to pass but not double stranded DNA        (dsDNA), e.g., a diameter of about 2 nm.    -   1.28. Any foregoing method wherein the monomer is a 3′-protected        nucleotide, e.g., deoxynucleotide triphosphate (dNTP), e.g.        selected from deoxyadenosine triphosphate (dATP), deoxyguanosine        triphosphate (dGTP), deoxycytidine triphosphate (dCTP),        deoxythymidine triphosphate (dTTP), for example dATP or dCTP.    -   1.29. Any foregoing method wherein the polymer is a nucleic acid        and the addition of the nucleotide to the nucleic acid is        catalyzed by a polymerase, e.g., a template independent        polymerase, e.g., terminal deoxynucleotidyl transferase (TdT),        or polynucleotide phosphorylase, e.g., wherein the polymerase        catalyzes the incorporation of a deoxynucleotide at the        3′-hydroxyl terminus of DNA.    -   1.30. Any foregoing method wherein the membrane contains a        multiplicity of nanopores and a multiplicity of polymers each        bound to a surface proximate to a nanopore, e.g., a multiplicity        of nucleic acids each bound via their 5′ end to a surface        proximate to a nanopore.    -   1.31. Any foregoing method wherein a multiplicity of polymers        each bound to a surface proximate to a nanopore, e.g., a        multiplicity of nucleic acids each bound at the 5′ end to a        surface proximate to a nanopore, are synthesized independently,        wherein each nanopore has an associated pair of electrodes,        wherein one electrode in the pair is located proximate to one        end of the nanopore and the other electrode located proximate to        the other end of the nanopore, such that each polymer can be        independently moved between the first and second chamber by        current provided by the pair of electrodes.    -   1.32. Any foregoing method wherein the polymer is a 3′-protected        nucleic acid bound at the 5′ end to a surface proximate to a        nanopore and the 3′ end of the 3′-protected nucleic acid is        drawn through the nanopore by using an electrical force, e.g.,        by using an electrical force applied from an electrode in an        adjacent chamber.    -   1.33. Method 1.20 wherein the new 3′-protected dNTP is the same        or different from the first 3′-protected dNTP.    -   1.34. Method 1.20 wherein the 3′-protected dNTP used in step (i)        of the cycle alternates with each cycle between 3′-protected        dATP and 3′-protected dCTP.    -   1.35. Any foregoing method wherein the polymer is a nucleic acid        and deprotection of the nucleic acid is carried out by an enzyme        that removes a 3′-protecting group on ssDNA but not on a        3′protected dNTP.    -   1.36. Any foregoing method further comprising the step of        detecting the sequence of the polymer as it passes through a        nanopore to confirm that the desired sequence has been        synthesized.    -   1.37. Any foregoing method comprising the step of detecting the        sequence of the polymer as it passes through a nanopore to        confirm that the desired sequence has been synthesized by        measuring the measuring the capacitive variance in a resonant RF        circuit as the DNA is drawn through the nanopore.    -   1.38. Any foregoing method wherein the reagents for addition of        one or more monomers or oligomers to the charged polymer        comprise reagents selected from a topoisomerase, a DNA        polymerase, or combinations thereof.    -   1.39. Any foregoing method wherein the addition of one or more        monomers or oligomers to the charged polymer is carried out        according to any of Method 2, et seq or Method A et seq.

For example, the invention provides a method for synthesizing a nucleicacid in a nanochip, comprising at least a first chamber and a secondchamber separated by a membrane comprising at least one nanopore, thesynthesis being carried out in a buffer solution by a cycle ofnucleotide addition to a first end of a nucleic acid having a first endand a second end, wherein the first end of the nucleic acid is moved byelectrical attraction between one or more addition chambers (whichcontains reagents capable of adding nucleotides) and one or more reservechambers (which do not contain reagents necessary to add nucleotides),the chambers being separated by one or more membranes each comprisingone or more nanopores, wherein the nanopore is large enough to permitpassage of the nucleic acid but is too small to permit passage of atleast one reagent essential for adding a nucleotide, e.g, wherein themethod corresponds to any of Method 1, et seq.

For example, Method 1A, which is a method, e.g., according to any ofMethod 1, et seq., for synthesizing a charged polymer comprising atleast two distinct monomers or oligomers in a nanopore-based device, thenanopore-based device comprising

-   -   one or more addition chambers or channels containing buffer        solution and reagents for addition of one or more monomers or        oligomers to the charged polymer in blocked form, such that only        a single monomer or oligomer can be added in one reaction cycle;        and    -   one or more deblocking chambers or channels containing buffer        solution and deblocking reagents for removing the blocker group        from the one or more monomers or oligomers added to the charged        polymer in blocked form,    -   wherein the addition chambers or channels are separated from the        deblocking chambers by one or more membranes comprising one or        more nanopores, and    -   wherein the charged polymer can pass through a nanopore and at        least one of the reagents for addition of one or more monomers        or oligomers cannot pass through a nanopore, and at least one of        the deblocking reagents cannot pass through a nanopore,    -   the method comprising    -   a. moving the first end of a charged polymer having a first end        and a second end, by electrical attraction, into an addition        chamber or channel, whereby monomers or oligomers are added to        said first end in blocked form,    -   b. moving the first end of the charged polymer with the added        monomer or oligomer in blocked form into a reserve chamber,        whereby the blocking group on the added monomer or oligomer is        removed, and    -   c. repeating steps a and b, wherein the monomers or oligomers        added in step a) are the same or different, until the desired        polymer sequence is obtained;        e.g., wherein the device comprises one or more first addition        chambers or channels containing reagents suitable for adding a        first type of monomer or oligomer and one or more second        addition chambers containing reagents suitable for adding a        second different type of monomer or oligomer, and wherein in        step a, the first end of the charged polymer is moved into        either the first addition chamber or the second addition        chamber, depending on whether it is desired to add a first type        of monomer or oligomer or a second different type of monomer or        oligomer.

In certain embodiments, the sequence of the polymer corresponds to abinary code, for example where the polymer is a nucleic acid and thesequence corresponds to a binary code, where each bit (0 or 1) isrepresented by a base, e.g. A or C.

In certain embodiments, the polymer is DNA.

In certain other embodiments, each bit is represented by a shortsequence of monomers rather than by a single monomer. For example, inone such embodiment, blocks of DNA are synthesized, where each blockgenerates a unique signal via the nanopore and corresponds to a zero ora one. This embodiment has certain advantages in that single nucleotidesare more difficult to detect in nanopores, especially solid-statenanopores, so using blocks is less prone to reading errors, although theinformation density in the polymer is correspondingly reduced.

For example, blocks of (double stranded) nucleotides can be added, usingsite-specific recombinases, i.e., enzymes that spontaneously recognizeand cleave at least one strand of a double strand of nucleic acidswithin a sequence segment known as the site-specific recombinationsequence. In one such embodiment, the site specific recombinase is atopoisomerase used to ligate a topo-conjugated dsDNA oligonucleotideblock to the sequence. These oligonucleotides themselves will not have astructure compatible with further ligation until they are cleaved with arestriction enzyme. Vaccinia virus topoisomerase I specificallyrecognises DNA sequence 5′-(C/T)CCTT-3′. The topoisomerase binds todouble-stranded DNA and cleaves it at the 5′-(C/T)CCTT-3′ cleavage site.Note that the cleavage is not complete, as the topoisomerase onlycleaves the DNA on one strand (although having a nearby nick on theother strand does cause a double-strand break of sorts), and when itcleaves, the topoisomerase attaches covalently to the 3′ phosphate ofthe 3′ nucleotide. The enzyme then remains covalently bound to the 3′end of the DNA, and can either religate the covalently held strand atthe same bond as originally cleaved (as occurs during DNA relaxation),or it can religate to a heterologous acceptor DNA having compatibleoverhangs, creating a recombinant molecule. In this embodiment, wecreate dsDNA donor oligonucleotides (e.g., comprising one of at leasttwo different sequences, one for ‘0’ and the other for ‘1’) flanked by atopoisomerase recombination site and a restriction site that generates atopoisomerase ligation site. The cassettes are Topo-charged; that is,they are covalently bound to a topoisomerase, which will bind them to atopoisomerase ligation site on the receiver oligonucleotide. When thegrowing DNA chain of the receiver is cleaved with a restriction enzymeit becomes capable of ligation to a Topo-charged cassette. So, one justneeds to cycle the growing DNA from restriction enzyme to Topo-chargedcassette successively, with each cycle adding another donoroligonucleotide. A related approach has been described for cloning, see,e.g., Shuman S., Novel approach to molecular cloning and polynucleotidesynthesis using vaccinia DNA topoisomerase. J Biol Chem. (1994);269(51):32678-84, the contents of which are incorporated by reference.

Single bases can be added using a similar strategy. In the presence of asuitable single stranded ‘deprotected’ ‘acceptor’ DNA, the topo-chargedDNA is enzymatically and covalently ligated (‘added’) to the acceptor bythe topoisomerase, which in the process becomes removed from the DNA. Atype IIS restriction enzyme can then cleave all of the added DNA withthe exception of a single base (the base which is being ‘added’). Thisprocess of deprotect-add can be repeated to add additional bases (bits).As demonstrated in the examples herein, it is feasible to use aTopo/TypeIIS restriction enzyme combination to add a single nucleotideto the 5′ end of a target single stranded DNA. The use of a TypeIISrestriction enzyme enables cleavage of DNA in a location different fromthat of the recognition sequence (other TypeIIS restriction enzymes canbe found athttps://www.neb.com/tools-and-resources/selectioncharts/type-iis-restriction-enzymes).The use of inosines (which act as ‘universal bases’ and pair with anyother base) in this system allows this reaction to occur without anyspecific sequence requirements in the target DNA. The identity of thenucleotide added to the single strand target DNA is the 3′ nucleotide towhich vaccinia topoisomerase conjugates via the 3′ phosphate. Since therecognition sequence of vaccinia topoisomerase is (C/T)CCTT, we haveused this system to add a ‘T’ to the target DNA. There is a relatedtopoisomerase, SVF, that can use the recognition sequence CCCTG(https://www.ncbi.nlm.nih.gov/pubmed/8661446). Thus SVF can be used toadd a ‘G’ instead of a ‘T’. Paired with vaccinia topo, binary data canbe encoded in T's and G's.

In another approach to single base addition, a 5′phosphate provides ablocking group to provide single base addition in the 3′ to 5′direction. The charging reaction charges the topoisomerase with a singleT (or G, or other nucleotide as desired), having a 5′ phosphate group.When the charged topoisomerase ‘sees’ a free 5′ unblocked(unphosphorylated) single stranded DNA chain it will add the T to thatchain, providing a DNA with a T added to the 5′. This addition isfacilitated by the presence of an adapter DNA having sequences to whichthe topoisomerase and the single stranded acceptor DNA can bind. (Notethat the adapter DNA is catalytic—it can be reused as a template inrepeated reactions.) The added nucleotide has a 5′ phosphate on it, soit won't be a substrate for further addition until it is exposed to aphosphatase, which removes the 5′ phosphate. The process is repeated,using vaccinia topoisomerase to add a single “T” to the 5′ end of atarget single stranded DNA and SVF topoisomerase to add a single ‘G’,thus allowing construction of a sequence encoding binary informationwith T and G. Other topoisomerases can be used to add A's or C's,although this reaction is less efficient.

When the topoisomerase is charged, there is a mix of charged anduncharged product, which represents an equilibrium between the twospecies. The ‘overhang’ that the topoisomerase leaves can be designed inmany ways, to optimize the efficiency of the reaction. Overhangs thatare rich in GC tend to have faster charging reactions, but have chargingequilibriums that tend to generate lower yield of product. We have foundthat having some base mismatches (or using inosines) instead of the‘proper’ pairs decreases the ‘reverse’ reaction and improves yield.Also, performing the reaction in the presence of polynucleotide kinase(plus ATP) improves yield by phosphorylating the reaction ‘byproduct’which decreases the reverse reaction rate. In certain embodiments, thetopoisomerase enzymes can be “bulked up” by adding additional amino acidsequences that do not impair function, so as to ensure that they arelarge enough that they cannot pass through the nanopore.

One advantage of using a topoisomerase-mediated strategy is that themonomer is covalently attached to the topoisomerase, and thereforecannot “escape” to interfere with other reactions. When polymerase isused, the monomers can diffuse so the polymerases and/or the deblockingagents should be specific (e.g. selective for A vs C, for example) oralternatively, the monomers are provided by a flow so they don't have achance to mix.

In one aspect, the invention provides a topoisomerase charged with asingle nucleotide, i.e., a topoisomerase conjugated to a singlenucleotide, e.g., wherein the topoisomerase is conjugated via the3′-phosphate of the nucleotide, and the nucleotide is protected, e.g.,phosphorylated, at the 5′-position.

In another aspect the invention provides a method (Method A) ofsynthesizing a DNA molecule using topoisomerase-mediated ligation, byadding single nucleotides or oligomers to a DNA strand in the 3′ to 5′direction, comprising (i) reacting a DNA molecule with a topoisomerasecharged with the desired nucleotide or oligomer wherein the nucleotideor oligomer is blocked from further addition at the 5′ end, then (ii)deblocking the 5′ end of the DNA thus formed, and repeating steps (i)and (ii) until the desired nucleotide sequence is obtained, e.g.,

-   -   A1.1. Method A which is a method of synthesizing a DNA molecule        by adding single nucleotides in the 3′ to 5′ direction        comprising (i) reacting a DNA molecule with a topoisomerase        charged with the desired nucleotide in 5′protected form, e.g.,        5′phosphorylated form, such that the desired nucleotide in        5′protected form is added to the 5′ end of the DNA, then (ii)        deprotecting the 5′ end of the DNA thus formed through the use        of a phosphatase enzyme, and repeating steps (i) and (ii) until        the desired nucleotide sequence is obtained; or    -   A1.2. Method A which is a method of synthesizing a DNA molecule        by adding oligomers in the 3′ to 5′ direction comprising (i)        reacting a DNA molecule with a topoisomerase charged with the        desired oligomer, thereby ligating the oligomer to the DNA        molecule, then (ii) using a restriction enzyme to provide a 5′        site for a topoisomerase-mediated ligation for another oligomer,        and repeating steps (i) and (ii) until the desired oligomer        sequence is obtained.    -   A1.3. Any foregoing method comprising providing ligase and ATP        to seal nicks in the DNA [NB: the topoisomerase ligation only        ligates one strand].    -   A1.4. Any foregoing method wherein the topoisomerase-charged        donor oligonucleotide comprises a 5′ overhang on the strand        complementary to the strand bearing the topoisomerase,        comprising a polyinosine sequence [NB: inosines act as        ‘universal bases’ and pair with any other base].    -   A1.5. Any foregoing method wherein the restriction enzyme is a        type IIS restriction enzyme which can cleave all of the added        DNA with the exception of a single base (the base which is being        ‘added’).    -   A1.6. Any foregoing method wherein the topoisomerase is selected        from vaccinia topoisomerase and SVF topoisomerase I.    -   A1.7. Any foregoing method wherein vaccinia topoisomerase (which        recognizes (C/T)CCTT) is used to add dTTP nucleotides and SVF        topoisomerase I (which recognizes CCCTG) is used to add dGTP        nucleotides, e.g., to provide binary code    -   A1.8. Any foregoing method wherein the DNA is double stranded        and the reserve chamber further comprises a ligase and ATP, to        repair the DNA strand not joined by the topoisomerase.    -   A1.9. Any foregoing method comprising use of a topoisomerase        inhibitor to suppress binding and activity of free topoisomerase        to the DNA oligomer, e.g., wherein the inhibitors is selected        from novobiocin and coumermycin.    -   A1.10. Any foregoing method wherein the DNA strand thus provided        has a sequence comprising thymidine (T) nucleosides and        deoxyguanosine (G) nucleosides.    -   A1.11. Any foregoing method wherein the topoisomerase adds a        single base, but the restriction enzyme cleaves at a position        which is one nucleotide in the 5′ direction from the base added        by topoisomerase.    -   A1.12. Any foregoing method wherein the DNA strand thus provided        has a sequence comprising a sequence of ‘TT’ and ‘TG’        dinucleotides.    -   A1.13. Any foregoing method wherein the DNA is single stranded,    -   A1.14. Any foregoing method wherein the DNA double stranded.    -   A1.15. Any foregoing method wherein the DNA is on a substrate or        magnetic bead, where it can be selectively exposed to or removed        from the reagents as required to provide the desired sequence.    -   A1.16. Any foregoing method wherein some or all of the reagents        for adding or deblocking the DNA are supplied by flow and        removed by flushing.    -   A1.17. Any foregoing method wherein the attachment of the single        nucleotides or oligomers to a single-stranded DNA is facilitated        by the presence of an adapter DNA having sequences to which the        topoisomerase and the single stranded acceptor DNA can bind.    -   A1.18. Any foregoing method carried out in a system where a        nanopore separates a chamber comprising the topoisomerase from a        chamber comprising the phosphatase or restriction enzyme,        wherein the nanopore allows movement of the DNA by electrical        attraction, but not the enzymes, e.g. as described in any of        Method 2, et seq.

One possible concern is poly-G sequences may form G-quartet secondarystructures. By moving the restriction enzyme back one base (to the 5′ ofthe topo sequence) and following a similar Topo/IIS strategy a ‘TT’ or‘TG’ can be added, each of which can represent a different bit. Whilethis would require 2 bases to encode a bit, it has the advantage ofavoiding poly-G sequences. In other embodiments, other bases in the 3′end of the topo recognition sequence—although less efficient than(C/T)CCTT, can allow conjugation using poxvirus topoisomerase with(C/T)CCTA, (C/T)CCTC and (C/T)CCTG(https://www.ncbi.nlm.nih.gov/pubmed/17462694). Proteinengineering/selection techniques can be used to improve the efficiencyof these reactions as well, and similar approaches can be used to addnon-canonical bases.

In certain embodiments, the method of synthesizing DNA by this methodincludes treating the DNA with a ligase and ATP. The topoisomerase onlyjoins together one side of the DNA (the other is essentially nicked).The ligase would repair the nick and ensure that the topoisomeraseitself doesn't recut the reaction product and cleave it.

In certain embodiments, the method comprises using a topoisomeraseinhibitor to suppress binding and activity of free topoisomerase to theDNA oligomer. Suitable inhibitors include novobiocin and coumermycin.Note that complete inhibition is not desirable, as a low level oftopoisomerase activity can help ‘relax’ coiled DNA, which is usefulespecially when synthesizing long DNA chains.

Thus, in another embodiment, the disclosure provides a method (Method 2)for synthesizing DNA in a nanochip, comprising one or more additionchambers containing a topoisomerase-charged oligonucleotide (i.e.oligonucleotide bound at the 3′ end to a topoisomerase), and one or morereserve chambers comprising a restriction enzyme or deblocker, e.g.,phosphatase, said chambers also containing compatible buffer solutionand being separated by a membrane comprising at least one nanopore,wherein the topoisomerase and the restriction enzyme are prevented frompassing through the nanopore (e.g. because they are too large and/orbecause they are tethered to a substrate in the first and secondchambers respectively), the synthesis being carried out by a cycle ofadding single nucleotides or short oligonucleotide blocks to a first endof a nucleic acid having a first end and a second end, wherein the firstend of the nucleic acid is moved by electrical attraction between anaddition chambers and a reserve chamber, for example in one embodimentas follows:

-   -   (i) moving the 5′ end of a receiver DNA (e.g., a double-stranded        DNA) into a first addition chamber, by means of an electrical        force,    -   (ii) providing in the first addition chamber a        topoisomerase-charged donor oligonucleotide, wherein the donor        oligonucleotide comprises a topoisomerase binding site, an        informational sequence (e.g., selected from at least two        different nucleotides or sequences, e.g., wherein one sequence        corresponds to ‘0’ and the other to ‘1’ in a binary code), and a        restriction site which when cleaved by a restriction enzyme will        yield a topoisomerase ligation site;    -   (iii) allowing sufficient time for the donor olignucleotide to        ligate to and thereby extend the receiver DNA;    -   (iv) moving the 5′ end of the receiver DNA thus extended into        the reserve chamber, by means of an electrical force, e.g., so        that the restriction enzyme cleaves the receiver DNA to provide        a topoisomerase ligation site, or in the case of single        nucleotide addition, the deblocker, e.g., phosphatase, generates        a 5′ unblocked nucleotide on the single stranded DNA; and    -   (v) repeating the cycle of steps (i)-(iv), adding        oligonucleotides having the same or different informational        sequence, until the desired DNA sequence or sequences are        obtained.

For example, the invention provides

-   -   2.1. Method 2 wherein the 3′ end of the receiver DNA is attached        proximate to a nanopore and the 5′end of the receiver        oligonucleotide comprises a topoisomerase ligation site, and        comprising a step after step (iv) of adding an additional        oligonucleotide to the 5′ end of the receiver DNA by flushing        the first addition chamber and providing new        topoisomerase-charged donor oligonucleotide to the first        addition chamber, wherein the new donor oligonucleotide has a        different informational sequence from the previous donor        oligonucleotide; and if desired that the new donor        oligonucleotide be added to the receiver DNA, drawing the 5′ end        of the receiver nucleic acid back into the first chamber, and        repeating steps (i)-(iii), or if not so desired, allowing the        receiver DNA to remain in the second chamber until the desired        donor oligonucleotide is provided to the first chamber.    -   2.2. Any foregoing method wherein a multiplicity of receiver DNA        molecules are synthesized independently in parallel, such that        DNA molecules having different sequences are obtained by        separately controlling whether they are present in the first        chamber.    -   2.3. Any foregoing method wherein a multiplicity of receiver DNA        molecules each bound at the 3′ end to a surface proximate to a        nanopore are synthesized independently, wherein each nanopore        has an associated pair of electrodes, wherein one electrode in        the pair is located proximate to one end of the nanopore and the        other electrode located proximate to the other end of the        nanopore, such that each receiver DNA molecule can be        independently moved between the first and second chamber by        current provided by the pair of electrodes.    -   2.4. Any foregoing method wherein the donor oligonucleotides        used in step (i) of the cycle alternate with each cycle between        donor oligonucleotides comprising a first informational sequence        and donor oligonucleotides comprising a second informational        sequence.    -   2.5. Method 2 comprising the step of adding an additional        oligonucleotide to the 5′ end of the receiver DNA by returning        the 5′ end of the receiver DNA to the first addition chamber to        add an oligonucleotide having the same informational sequence or        moving the 5′ end of the receiver DNA to a second addition        chamber to having a donor oligonucleotide bound at the 3′ end to        a topoisomerase, wherein the donor oligonucleotide in the second        addition chamber has a different informational sequence from the        donor oligonucleotide in the first addition chamber.    -   2.6. Any foregoing method wherein the donor oligonucleotide        comprises a structure as follows:

(SEQ ID NO 1) 5′ CGAAGGG <Informational sequence A or B> GTCGACN NNNN3′ GCTTCCC <---------Complement----------> CAGCTGN NNNN

-   -   wherein N refers to any nucleotide and the restriction enzyme is        Acc1, which can cut the DNA (e.g. GTCGAC in the above sequence)        so as to provide an appropriate overhang.    -   2.7. Any foregoing method wherein the donor oligonucleotide has        a hairpin structure, e.g., 2.6 wherein the NNNNN groups on the        top and bottom strands are joined.    -   2.8. Any foregoing method wherein at least one of the        topoisomerase charged oligonucleotides has a structure as        follows:

(SEQ ID NO 1) 5′ CGAAGGG <Informational sequence A or B> GTCGACN NNNN3′  *TTCCC <---------Complement----------> CAGCTGN NNNN (*= topoisomerase)

-   -   2.9. Any foregoing method wherein at least one of the        topoisomerase charged oligonucleotides has a structure as        follows:

5′ pCACGTCAGGCGTATCCATCCCTT* 3′  GTGCAGTCCGCATAGGTAGGGAAGCGC

-   -   2.10. The preceding method wherein the topoisomerase charged        oligonucleotide    -   2.11. Any foregoing method wherein the sequence of DNA        synthesized is determined following each cycle by detecting the        change in electric potential, current, resistance, capacitance        and/or impedance as the oligonucleotide passes through the        nanopore.    -   2.12. Any foregoing method wherein the synthesis of the DNA        takes place in a buffer solution, e.g., a solution comprising a        buffer for pH 7-8.5, e.g. ca. pH 8, e,g, a buffer comprising        tris(hydroxymethyl)aminomethane (Tris), a suitable acid, and        optionally a chelater, e.g., ethylenediaminetetraacetic acid        (EDTA), for example TAE buffer containing a mixture of Tris        base, acetic acid and EDTA or TBE buffer comprising a mixture of        Tris base, boric acid and EDTA; for example a solution        comprising 10 mM Tris pH 8, 1 mM EDTA, 150 mM KCl, or for        example, 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM        Magnesium Acetate, pH 7.9 @ 25° C.    -   2.13. Any foregoing method further comprising removing the DNA        from the nanochip.    -   2.14. Any foregoing method further comprising amplifying the DNA        thus synthesized.    -   2.15. Any foregoing method further comprising removing the DNA        from the nanochip and crystallizing the DNA.    -   2.16. Any foregoing method further comprising stabilizing the        DNA, e.g., by drying a solution comprising the DNA together with        one or more of a buffer (e.g., a borate buffer), an antioxidant,        a humectant, e.g. a polyol, and optionally a chelator, for        example as described in U.S. Pat. No. 8,283,165 B2, incorporated        herein by reference, or by forming a matrix between the nucleic        acid and a polymer, such as poly(ethylene glycol)-poly(l-lysine)        (PEG-PLL) AB type block copolymer.    -   2.17. Any foregoing method comprising providing ligase and ATP        to seal nicks in the DNA [NB: the topoisomerase ligation only        ligates one strand].    -   2.18. Any foregoing method wherein the topoisomerase-charged        donor oligonucleotide comprises a 5′ overhang on the strand        complementary to the strand bearing the topoisomerase,        comprising a polyinosine sequence [NB: inosines act as        ‘universal bases’ and pair with any other base].    -   2.19. Any foregoing method wherein the restriction enzyme is a        type IIS restriction enzyme which can cleave all of the added        DNA with the exception of a single base (the base which is being        ‘added’).    -   2.20. Any foregoing method wherein the topoisomerase is selected        from vaccinia topoisomerase and SVF topoisomerase I.    -   2.21. Any foregoing method wherein vaccinia topoisomerase (which        recognizes (C/T)CCTT) is used to add dTTP nucleotides and SVF        topoisomerase I (which recognizes CCCTG) is used to add dGTP        nucleotides, e.g., to provide binary code information.    -   2.22. Any foregoing method wherein the reserve chamber further        comprises a ligase and ATP, to repair the DNA strand not joined        by the topoisomerase.    -   2.23. Any foregoing method comprising use of a topoisomerase        inhibitor to suppress binding and activity of free topoisomerase        to the DNA oligomer, e.g., wherein the inhibitors is selected        from novobiocin and coumermycin.    -   2.24. Any foregoing method wherein the DNA strand thus provided        has a sequence comprising thymidine (T) nucleosides and        deoxyguanosine (G) nucleosides.    -   2.25. Any foregoing method wherein the topoisomerase adds a        single base, but the restriction enzyme cleaves at a position        which is one nucleotide in the 5′ direction from the base added        by topoisomerase.    -   2.26. Any foregoing method wherein the DNA strand thus provided        has a sequence comprising a sequence of ‘TT’ and ‘TG’        dinucleotides.    -   2.27. Any foregoing method which is a method of synthesizing a        DNA molecule by adding single nucleotides in the 3′ to 5′        direction comprising (i) reacting a DNA molecule with a        topoisomerase charged with the desired nucleotide in 5′protected        form, e.g., 5′phosphorylated form, such that the desired        nucleotide in 5′protected form is added to the 5′ end of the        DNA, then (ii) deprotecting the 5′ end of the DNA thus formed        through the use of a phosphatase enzyme, and repeating steps (i)        and (ii) until the desired nucleotide sequence is obtained.    -   2.28. Any foregoing method which is a method of synthesizing a        DNA molecule by adding oligomers in the 3′ to 5′ direction        comprising (i) reacting a DNA molecule with a topoisomerase        charged with the desired oligomer, thereby ligating the oligomer        to the DNA molecule, then (ii) using a restriction enzyme to        provide a 5′ site for a topoisomerase-mediated ligation for        another oligomer, and repeating steps (i) and (ii) until the        desired nucleotide sequence is obtained.    -   2.29. Any foregoing method which is a method in accordance with        any of Method A, et seq.

The product of the synthesis reactions can be detected, reviewed forquality control purposes, and read to extract the data encoded on thepolymer. For example the DNA may be amplified and sequenced byconventional means to confirm that the nanopore sequencing is robust.

In another embodiment, the invention provides an oligonucleotidecomprising a topoisomerase binding site, an informational sequence(e.g., selected from at least two different sequences, e.g., wherein onesequence corresponds to ‘0’ and the other to ‘1’ in a binary code), anda restriction site which when cleaved by a restriction enzyme will yielda topoisomerase ligation site, e.g., comprising the following sequence:

(SEQ ID NO 2) 5′ CGAAGGG <Informational sequence A or B> GTCGAC3′ GCTTCCC <---------Complement----------> CAGCTGwherein the Informational Sequence A or B is a sequence of 3-12, e.g.,about 8 nucleotides.

In another embodiment, the invention provides a topoisomerase chargedoligonucleotide wherein the oligonucleotide comprises a topoisomerasebinding site, an informational sequence (e.g., selected from at leasttwo different sequences, e.g., wherein one sequence corresponds to ‘0’and the other to ‘1’ in a binary code), and a restriction site whichwhen cleaved by a restriction enzyme will yield a topoisomerase ligationsite; for example a topoisomerase charged oligonucleotide having astructure as follows:

(SEQ ID NO 1) 5′ CGAAGGG <Informational sequence A or B> GTCGACN NNNN3′  *TTCCC <---------Complement----------> CAGCTGN NNNNwherein the Informational Sequence A or B is a sequence of 3-12, e.g.,about 8 nucleotides and * is topoisomerase covalently bound to theoligonucleotide; e.g., wherein the topoisomerase is Vaccinia virustopoisomerase I.

In certain embodiments, the nanopore chips are controlled to carry out amethod of synthesizing and/or reading the polymer, e.g., in accordancewith any of Methods 1, et seq., Method A, et seq., or Method 2, et seq.,using a computerized chip controller.

For example, referring to FIG. 65, a partial perspective drawing havingselective transparent surfaces of a grouping of 3-chamber nanopore-basedcells 6500 (each cell similar to that discussed herein above), ofnanopore memory chip is shown for some embodiments of the presentdisclosure. In particular, a group of four 3-chamber cells6506,6508,6510,6512 are connected together, such that the upper (or top)left chambers 6502 (Add “0” chambers) of each of the connected cells6506-6512 are fluidically connected together to form an Add “0” flowchannel or Add “0” chambers 6502. In addition, the upper (or top) rightchambers 6504 (Add “1” chambers) of each of the connected cells6506-6512 are also fluidically connected together to form a separate Add“1” flow channel or Add “1” chambers 6504. In addition, the Add “0”chambers (or channel) 6502 have a common electrode 6520, and the Add “1”chambers (or channel) 6504 have a different common electrode 6522. Insome embodiments there may be a single metallic or conductive stripproviding the common electrode for each add channel, and in someembodiments there may be separate electrodes, which are connected byin-chip wiring.

Below the collective Add channels 6502,6504, are individual “deblock”chambers 6530-6536, similar to that discussed herein above, that areboth fluidically and electrically isolated from the other chambers. Onthe bottom of each of the deblock chambers 6530-6536 are correspondingindividually controllable “deblock” electrodes, e.g., deblock electrodes6514,6516 visible in FIG. 65 correspond to deblock chambers 6534,6536,respectively. Also, the upper chambers for the cells 6506-6512 each havea corresponding nanopore 6528 through a membrane 6529. Also, in thisexample, the fluidic cell 6512 has a left top Add “0” chamber 6537 and aright top Add “1” chamber 6539. While the Add “0” chambers for thefluidic cells 6502-6512 are fluidically connected via the fluidicchannels 6502, and the Add “1” chambers for the fluidic cells 6502-6512are fluidically connected via the fluidic channels 6504, each of thefluidic cells 6506-6512 has an independent memory storage string (e.g.,DNA or polymer) 6550, which has one end that traverses through thenanopore 6550 to enter the Add “1” or Add “0” chambers, and returns toits corresponding deblock chamber 6530-6536, which is fluidically andelectrically isolated from the other chambers (in this example). Thus,each of the 3-chamber fluidic cells 6506-6512 represents an independentmemory storage cell, or memory cell (discussed more hereinafter).

As the configuration of FIG. 65 has all the Add “0” electrodes connectedtogether and, separately, all the Add “1” electrodes connected together,and the deblock electrodes are individually controlled, the writing (oradding) may occur in write (or add) “cycles,” such as an Add “0” cycle,when all the cells that need to write a “0” may be written at the sametime, followed by an Add “1” cycle, when all the cells that need towrite a “1” may all be written at the same time. Other data writingcycles or approaches may be used if desired.

In addition, the Add “0” and Add “1” channels 6502, 6504, may be filledwith fluid (or flushed, or washed or emptied) from the front or back, asshown by the arrows 6503-6505, respectively, and the deblock chambers6530-6536 may be filled with fluid (or flushed, or washed or emptied)from the side, as shown by the arrows 6540-6546, respectively. It is notrequired that every Add “1” chamber be fluidically and electricallyconnected or that every Add “0” chamber be fluidically and electricallyconnected. If a large number of them are so connected it providesefficiencies; in general, the more cells that are connected the moreefficiencies that can be realized.

Also, the entire polymer (or DNA) or “string” or memory string 6550 maybe prevented from completely exiting the central deblock chamber bybinding (or tethering or attaching) one end of the polymer 6550 to thesurface of the central deblock chamber 6536, e.g., shown as point 6552in deblock chamber 6536. Other locations in deblock chamber 6536 may beused to tether the polymer provided it meets the desired functional andperformance requirements. In some embodiments, a structure 6554, e.g., abead, particle, or origami, or other structure, may be attached to oneend of the polymer 6550 and prevent the polymer from leaving the deblockchamber 6536 through the nanopore 6550. Similar criteria apply for thepolymer memory string 6550 in the other deblock chambers 6530-6534.

The polymer 6550 used to store the data may be DNA as discussed herein,or it may be any other polymer or other material that has the propertiesdescribed herein. The polymer 6550 used to store data may also bereferred to herein as a “memory polymer” or “memory string” (due to itsstring-like appearance).

Referring to FIG. 66, a partial perspective drawing having selectivetransparent surfaces of a grouping of 3-chamber nanopore-based cells6600 (each cell similar to that discussed herein above), of nanoporememory chip is shown for some embodiments of the present disclosure. Inparticular, similar to FIG. 65, a group of four 3-chamber cells6606,6608,6610,6612 are connected together, such that the upper (or top)left chambers 6602 (Add “0” chambers) of each of the connected cells6606-6612 are fluidically connected together to form an Add “0” flowchannel 6602. In addition, the upper (or top) right chambers 6604 (Add“1” chambers) of each of the connected cells 6606-6612 are alsofluidically connected together to form a separate Add “1” flow channel6604. However, in this embodiment, the Add “0” chambers associated withthe cells 6606-6612 have separate electrodes 6620-6626, and the Add “1”chambers associated with the cells 6606-6612 also have separateelectrodes 6630-6636. This fluidic and electrode arrangement is similarto that described and shown herein above with FIG. 27. In someembodiments, the upper chambers (Add “0” and Add “1”) may be fluidicallyseparated or isolated from each other to avoid potentialelectrical-cross talk between adjacent Add chambers when trying tocontrol the path of the DNA.

Also, for FIG. 65, deblock chambers may be fluidically connected eventhough the electrodes are separately controlled. In that case, there maybe cross-talk between the channels, e.g., nearby DNA gets attracted byelectric fields and/or current flow seen by adjacent cells.

In some embodiments, the electrodes may be have 3D shapes, such as atriangle or pyramid rising up from the bottom of the cell or protrudingdown into the cell. In that case, the electrode may be constructed toproduce a more targeted, focused or closer electric field to thenanopore for that cell, which may reduce cross talk between adjacentcells that are fluidically connected but electrically separated.

If the memory string (or DNA or polymer) gets so long it may wrap aroundfrom one add chamber and though the top of another. To avoid that issue,there may be partial walls disposed between adjacent cells along theflow channel, to make the distance between adjacent nanopores that muchlonger for long DNA.

Below the Add chambers is a common “deblock” chamber 6640, which iscommon to all the upper Add chambers, similar to that discussed hereinabove. On the bottom of the common deblock chamber 6540 is a commondeblock electrode 6642. Also, the upper chambers for the cells 6606-6612may each have the nanopore 6528, similar to that discussed herein above,through the membrane 6529.

In addition, the deblock chamber 6540 may be filled with fluid from aside (depending on the structural configuration of the cell). In someembodiments, it may be filled from the left (or right side), as shown bythe arrow 6550. In other embodiments, it may be filled from the front(or back) side, as shown by the arrow 6652.

Also, the entire DNA or polymer “string” (or memory string) 6550 may bekept from completely exiting the central deblock chamber by binding (ortethering) one end of the polymer 6550 to the surface of the centraldeblock chamber 6640, e.g., shown as point 6552 for the cell 6612. Asimilar arrangement would apply for the other cells 6606-6610. Otherlocations may be used to tether the polymer provided it meets thedesired functional and performance requirements.

Referring to FIG. 67, a schematic circuit block diagram of ananopore-based “memory chip” 6700, is shown for embodiments of thepresent disclosure. In particular, the memory chip 6700 may have aplurality of nanopore-based “memory cells” 6702 (or “storage cell” or“data storage cell”), each having the ability to store data. Each of the“memory cells” 6702 has a multi-chamber nanopore-based fluidic cell 6704with a cell structure similar to that discussed herein above (e.g.,having a membrane with a nanopore and the “memory string” 6550 (e.g.,DNA or other polymer, as discussed herein). The “memory cells” 6702 mayalso include any solid-state or semiconductor passive or activecircuitry or chip layers or components, which interface with the fluidiccell 6704 to provide the data storage (or writing or adding) and/or dataretrieval (or reading or sequencing) functions described herein.

The memory cells 6702 may be connected together (electrically andfluidically), such as 3-chamber cells having common fluidic “Add”channels and common “Add” electrodes, and independent “deblock”chambers, such as is shown and described with FIG. 65. Any number ofchambers and any cell configurations described herein may be used ifdesired.

The “memory cells” 6702 may be configured as an M×N array, with M rowsand N columns, each of the cells 6702 being labeled C_(M,N). Morespecifically, the cells 6702 in the first row are labeledC_(1,1)-C_(1,N), and the cells 6702 in the last row are labeledC_(M,1)-C_(M,N). M and N may be any values that provide the desiredfunctions and performance, and M,N may each be as small a 1 and as largeas 1 million, 10 million, 100 million, 1 billion, or 1 trillion, orlarger, depending on the desired footprint size of the memory chip andthe size of each memory cell.

The memory chip 6700 has an Add “0” input DC voltage on line 6710, whichis electrically connected (directly or through on-chip circuitry orcomponents, as described herein) to each of the Add “0” electrodes. TheAdd “0” input DC voltage on the line 6710 drives the Add “0” electrodeto the desired voltage state (discussed herein), to help position (ormove or steer) the memory string 6550 (DNA or other polymer, asdiscussed herein) to the desired chamber of the fluidic cell 6704. Inthis configuration, all the Add “0” electrodes for each of the memorycells are shared or common, or electrically connected, as shown in FIG.65.

The memory chip 6700 also has an Add “1” input DC voltage on line 6712which is electrically connected (directly or through on-chip circuitryor components as described herein) to each of the Add “1” electrodes.The Add “1” input DC voltage on the line 6710 drives the Add “1”electrode to the desired voltage state (discussed herein), to helpposition (or move or steer) the memory string 6550 (DNA or otherpolymer, as discussed herein) to the desired chamber of the fluidic cell6704. In this configuration, all the Add “1” electrodes 6522 for each ofthe memory cells is shared or common, as shown in FIG. 65.

The memory chip 6700 also has a “Deblock” input DC voltage on aplurality of lines (or bus) 6714, each of which is electricallyconnected (directly or through on-chip circuitry or components asdescribed herein) to a corresponding “deblock” electrode in each of thecells 6702. The deblock input DC voltage drives the correspondingdeblock electrode for a given cell to the desired voltage state(discussed herein), to help position (or move or steer) the memorystring 6550 (DNA or other polymer, as discussed herein) to the desiredchamber of the fluidic cell 6704. In this configuration, each of thedeblock electrodes are independently driven, as shown in FIG. 65, thusthe need for the plurality of electrical connections or bus (or deblockbus) 6714. Each row of memory cells 6702 will have a correspondingnumber of deblock input DC voltage lines provided. For example, thefirst row there is a set of N deblock lines 6716 feeding the N cells6702 in that row, and in the last row M, there is a separate set of Ndeblock lines 6718 feeding the N cells 6702 in the row M.

The DC input voltages Add “0”, Add “1”, and deblock, on the lines 6710,6712, 6714, respectively, may be referred to herein as DC “steering”voltages V_(ST) (or polymer or DNA steering voltages or memory stringsteering voltages) as they are used to “steer” the polymer memory stringto the appropriate chamber of the fluidic cell 6704 at the appropriatetime to achieve the desired result, e.g., write or add a “0” or “1” ontothe memory string, or do nothing, or move the memory string to aparticular chamber to enable writing or reading data, or performvalidation testing, or the like. DC input voltages Add “0”, Add “1”, anddeblock, on the lines 6710, 6712, 6714, respectively, may be providedfrom a computer-based controller circuit or logic or device, asdescribed herein, which has the appropriate logic to perform thefunctions described herein.

The memory chip 6700 also has an AC input voltage Vin, and an AC outputvoltage Vout, on line 6720, 6722, respectively. The AC input voltage Vinon the line 6720 is electrically connected, as described herein, to eachof the memory cells 6702 in parallel. The AC Vin provides an AC signal,e.g., rf or radio frequency signal, on the line 6714 to each of thememory cells 6702 and the memory cells are configured to be a resonatoror nanopore polymer resonator (NPR), each having a different frequencyresponse to the input AC Vin, as discussed herein. The line 67 mayconnect the memory cell 6702 and/or the electronic components on thechip, the electrodes, and the fluidic cell 6704 therein, differentlyfrom that shown in FIG. 67, depending on the circuit configuration usedfor the nanopore polymer resonator (NPR), fluidic cell configuration,electrode configuration, or other factors, as described herein. The ACinput voltage Vin on the line 6520 may be provided from a computer-basedcontroller circuit or logic or device, as described herein, which hasthe appropriate logic to provide the appropriate AC input voltage Vinand perform the functions described herein.

The combined frequency response from each of the memory cell 6702 may beprovided to an on-chip amplifier (or pre-amp) 5320 (FIG. 53), whichprovides the AC output voltage Vout on the line 6722 indicative of thecombined frequency response. The AC output voltage Vout on the line 6722may be provided to a computer-based processing circuit or logic ordevice, which has the appropriate logic, e.g., analog-to-digital (A/D)conversion and digital signal processing (DSP) logic, as describedherein, which reads the data stored on the memory string 6550 and mayperform other functions as described herein.

Referring to FIG. 68, a top level hardware block diagram is shown of aread/write memory storage system 6800 having the nanopore-based memorychip 6700 (FIG. 67) and a memory read/write controller 6802, inaccordance with embodiments of the present disclosure. In particular,the memory read/write controller 6802 may have a write controller logic6804, which receives input data to be written to the memory chip 6700 onlines and an address to store the data (or label or pointer or the like)on lines 6808, and provides the DC steering voltages Add “0”, Add “1”,and deblock, on the lines 6710, 6712, 6714, respectively, to thenanopore memory chip 6700. The write controller 6804 has the appropriatehardware, software and firmware (including any microprocessor ormicro-computer based processor chips or devices and/or memory storage)as needed to provide the functions described herein, as indicated by abox 6810.

In addition, the write controller 6804 may also provide a write (or add)cycle clock 6812 (or oscillator), which determines when the memory chip6700 writes (or adds or stores) “0” or “1” bits. In particular, thewrite controller chip 6804 provides the DC steering voltages (Add “0”,Add “1”, Deblock) based on the write cycle clock 6812 to cause thememory chip 6700 to write “1” or “0” to the memory cells. As discussedherein above with FIG. 65, in certain cell configurations, such as whenall the Add “0” electrodes are connected together and, separately, allthe Add “1” electrodes are connected together, and the deblockelectrodes are individually controlled (such as in FIG. 65), the writing(or adding) of data bits may occur in write (or add) “cycles,” such asan Add “0” cycle, when all the cells that need to write a “0” may bewritten at the same time, followed by an Add “1” cycle, when all thecells that need to write a “1” may all be written at the same time. Thewrite cycle clock provides a write cycle signal on a line 6814 to enablethe write requesting device or platform or computer bus, to determinethe writing status of the memory chip. Other data writing cycles,timing, or approaches may be used if desired.

In some embodiments, the write controller 6802 may also receive controlsignals from the system or computer bus, such as a Write Request (W-REQ)signal on a line 6820 to request certain data be written to the memorychip 6700, and the write controller 6802 may also provide a Write (orAdd) Complete (W-COM) signal on a line 6822, to indicate when therequested data has been written to the memory chip 6700.

The memory read/write controller 6802 may also have memory readcontroller logic 6850, which may receive a read address (or label orpointer or the like) on lines 6852 corresponding to the storage locationof the data desired to be read from the memory chip 6700, and providesthe requested data read from the memory chip 6700, on the lines 6854.The read controller 6850 may also have the necessary logics andcomponents to provide the AC input voltage signal Vin to the memory chip6700 on the line 6720. The AC input voltage Vin, as described herein, isan AC rf (radio frequency) signal that has frequency componentscorresponding to the bandwidth of the nanopore resonators (NPRs) in thememory chip 6700. To provide the Vin signal, the read controller 6850may have a frequency oscillator logic 6858 (programmable ornon-programmable), which provides the necessary frequency components(discussed herein) to enable the read controller logic to read therequested data from the nanopore memory chip 6700. As discussed herein,the AC Vin signal may be directly synthesized, combine multiple probefrequencies, and may be a single broadband signal, or a time swept orstepped frequency signal, or any other AC signal the provides thefunctions described herein.

The read controller 6850 also receives the output AC Vout voltage fromthe memory chip 6700 on the line 6722, and performs A/D conversion anddigital signal processing (e.g., using on-board A/D conversion logic6862 and FFT logic 6864), as discussed herein, on the Vout signal todetermine the values of the desired data at the specified read addressand provide the output data on Read Data Out the lines 6854.

The read controller 6850 has the appropriate hardware, software andfirmware (including any microprocessor or micro-computer based processorchips or devices and/or memory storage) as needed to provide thefunctions described herein, as indicated by a box 6856.

In addition, the read controller 6850 may also receive the write (oradd) cycle clock signal on the line 6814 from the write cycle clock 6812(or oscillator), which, as discussed above, determines when the memorychip 6700 will write (or add or store) “0” or “1” bits. In particular,controller chip 6804 will provide the DC steering voltages (Add “0”, Add“1”, Deblock) based on the write cycle clock 6812 to cause the memorychip 6700 to write “1” or “0” to the memory cells. Because the act ofwriting with the present disclosure requires the DNA (or polymer ormemory string) to pass through the nanopore to enter the desired chamberto Add bits and also to pass through the nanopore when exiting back tothe deblock chamber, the write cycle clock signal may also be used bythe read controller 6850 to determine when is the best time to read thedata, discussed more hereinafter with FIG. 69.

In some embodiments, the read controller may provide a read signal 6860to the write controller 6804 to request the controller 6804 provide thenecessary steering voltages (Add “0”, Add “1”, Deblock) on the lines6710-6714 to cause the memory string to pass through the nanopore toenable reading of the memory string.

In some embodiments, the read controller 6850 may also receive a ReadRequest (RD-REQ) signal on a line 6870 to request certain data be readfrom the memory chip 6700, and the read controller 6850 may also providea Read Complete (RD-COM) signal on a line 6822, to indicate when therequested data has been read from the memory chip 6700. The MemoryController 6802 may perform only one function, e.g., read or writing tothe nanopore chip if desired, or it may perform both of these functions(Read and Write), if desired.

Referring to FIG. 68A, the Nanopore memory system 6800 may be part of alarger computer system which may interact with an Address/Data/ControlBus 6870, and may also interact with separate memory controllers 6876,all of which interact with one or more CPU/Processors 6874. For example,one or more of the read/write address and/or data inputs, outputs and/orcontrol lines, such as numerals 6820, 6822, 6806, 6808, 6814, 6852,6854, 6872, 6870, shown in FIG. 68, may be received from or provided tothe bus 6872 or the memory controller 6876. The computer system 8670 mayinterface with a user 6878 and a display screen 6880.

Referring to FIG. 69, a table 6900 of sample DC steering voltages(V_(ST)) for the configuration shown in FIG. 65 and corresponding timegraphs 6902 for the steering voltages V_(ST) and associated results onthe memory chip 6700, in accordance with embodiments of the presentdisclosure, is shown. In particular, the table 6900 shows the DCsteering voltages V_(ST) (e.g., Add “0”, Add “1”, Deblock, or V_(ST0),V_(ST1), V_(STDB), respectively) to be provided to the respectiveelectrodes of the memory cell, based on the write cycle timing to causethe memory chip 6700 to write “1” or “0” to the memory cells, such as anAdd “0” cycle, when all the cells that need to write a “0” may bewritten at the same time, followed by an Add “1” cycle, when all thecells that need to write a “1” may all be written at the same time.

Referring to FIG. 69 and FIG. 65, sample steering voltages for the Add“1” cycle are shown in column 6906 (FIG. 69), and steering voltages forthe Add “0” cycle are shown in column 6908. More specifically, duringAdd “1” cycle, it is desired to cause the memory string (DNA or polymer)6550 to traverse through the nanopore 6528 to the Add “1” chamber 6539(FIG. 65) of the fluidic cell 6512. This may be done by having the Add“1” electrode voltage V_(ST1) be at ground (GND) or 0 Volts, the Add “0”electrode voltage V_(ST0) be at a negative voltage (relative to the Add“1” voltage), and the deblock electrode voltage V_(STDB) be at anegative voltage (relative to the Add “1” voltage), until the “1” bit iswritten (or added) to the string 6550. After the “1” bit is written, thestring 6550 may be pulled back into the deblock chamber 6536 (to beready for the next write command) by having the deblock voltage changeto a positive voltage (relative to the Add “1” voltage and the Add “0”voltage), which will attract the memory string if it has a net negativecharge, such as DNA.

The time plots 6902 show the values of the steering voltages for the Add“1” and Add “0” write cycles. In this case, for plot 6910, for the Add“1” cycle, shows the Add “1” voltage value is held at a constant valueof GND (0 volts) for the entire Add “1” cycle, and plot 6912 shows thevalue of the Add “0” voltage is held at a constant value of a negativevoltage (e.g., −1.0 volts) for the entire Add “1” cycle. The plot 6914for the “deblock” voltage shows a square wave having two parts 6916 and6920, which starts at a negative voltage value (as discussed hereinabove) to release the memory string from the deblock chamber 6536 (FIG.65) and allow one end of the memory string 6550 to traverse from thedeblock chamber 6536 through the nanopore 6528 to the Add “1” chamber6539, where the Add “1” bit reaction occurs as shown on the graph 6914,also indicated by a “W1” for write of a “1” bit. A first portion of thegraph section 6918 (labeled “T1”) is the time it takes for the memorystring (DNA or polymer) to traverse the nanopore 6528 into the Add “1”chamber 6539, after which the “addition” chemical reaction occurs,during the time “W1”. The amount of time W1 should be set long enoughfor the “1” bit addition reaction to complete, which may have a reactiontime of, e.g., about 0.01-100 Hz, or about 10 seconds to 100milliseconds. Other addition reaction times for the addition reactionsmay be used if desired, depending on the chemistry used, as discussedherein.

Next in the Deblock time graph section 6920, the Deblock voltage becomespositive relative to the Add “1” voltage, which pulls memory string 6550back through the nanopore 6528 into the Deblock chamber, after which itis held for a HOLD time period T_(H1) long enough for the deblockreaction to occur, as discussed herein (similar to the Add reactiontime). The time “T2” indicated by numeral 6922 is the time it takes forthe memory string 6550 to traverse the nanopore 6528. For the remainderof time (Hold time, T_(H1)) in this portion 1920 of the cycle the stringis held in the Deblock chamber, waiting for the next write request.Thus, other than the deblock reaction, there is no activity (NA)occurring on the string during this Hold time.

Next the write cycle repeats, this time for the Add “0” cycle, column6908. Referring to FIG. 69 and FIG. 65, sample steering voltages for theAdd “0” cycle are shown in column 6908. More specifically, during Add“0” cycle, it is desired to cause the memory string (DNA or polymer)6550 to traverse through the nanopore 6528 to the Add “0” chamber 6537(FIG. 65) of the fluidic cell 6512. This may be done by having the Add“0” electrode voltage V_(ST0) be at ground (GND) or 0 Volts, the Add “1”electrode voltage V_(ST1) be at a negative voltage (relative to the Add“0” voltage), and the Deblock electrode voltage V_(STDB) be at anegative voltage (relative to the Add “0” voltage), until the “0” bit iswritten (or added) to the string 6550. After the “0” bit is written, thestring 6550 may be pulled back into the deblock chamber 6536 (to beready for the next write command) by having the deblock voltage changeto a positive voltage (relative to both the Add “1” voltage and the Add“0” voltage), which will attract the memory string if the string has anet negative charge, such as DNA.

Similarly, for the Add “0” cycle, the plot 6912 for the Add “0” cycleshows the Add “0” voltage value is held at a constant value of GND (0volts) for the entire Add “0” cycle, and plot 6910 shows the value ofthe Add “0” voltage is held at a constant value of a negative voltage(e.g., −1.0 volts) for the entire Add “0” cycle. The plot 6914 for the“deblock” voltage for the Add “0” cycle shows a square wave having twoparts 6926 and 6930, which starts at a negative voltage value (asdiscussed herein above) to release the memory string from the deblockchamber 6536 (FIG. 65) and allow one end of the memory string 6550 totraverse from the deblock chamber 6536 through the nanopore 6528 to theAdd “0” chamber 6537, where the Add “0” bit reaction occurs as shown onthe graph 6914, also indicated by a “W0” for write bit “0”. A firstportion of the graph section 6924 (labeled “T3”) is the time it takesfor the memory string (DNA or polymer) to traverse the nanopore 6528into the Add “0” chamber 6537, after which the “addition” of bit “0”chemical reaction occurs, during the time “W0”. The amount of time W0should be set long enough for the addition reaction to complete, whichmay have a reaction time of, e.g., about 10-100 Hz, or about 10 to 100milliseconds. Other addition reaction times for the addition may be usedif desired, depending on the chemistry used, as discussed herein.

Next in the Deblock time graph section 6930, the Deblock voltage becomespositive relative to the Add “0” voltage, which pulls memory string 6550back through the nanopore 6528 into the Deblock chamber 6536, afterwhich it is held for a HOLD time period T_(H2) long enough for thedeblock reaction to occur, as discussed herein (similar to the Addreaction time). The time “T4” indicated by numeral 6928 is the time ittakes for the memory string 6550 to traverse the nanopore 6528 andreenter the Deblock chamber. For the remainder of time (Hold time,T_(H2)) in this portion 1930 of the write cycle, a deblock reactionoccurs (as discussed herein) on the string and the string is held in theDeblock chamber, waiting for the next write request. Thus, other thanthe deblock reaction, there is no activity (NA) occurring on the stringduring this Hold time.

Thus, for the embodiment described, the deblock voltage may control thewriting of a “1” or “0”, releasing of the memory string to enter thecorresponding Add chamber, and the removal of the memory string from thechamber after writing and the holding of string in the deblock chamber.Thus, the deblock voltage may create a No Activity (NA) state or donothing state, if desired for a given write cycle or portion thereof, byadjusting the “Hold” time during the cycle. It can also determine thetiming of when the write (or add) time begins and ends by adjusting thewrite times W1, W0 during the cycle. Also, depending on the traversetime T1-T4 it takes the memory string to fully traverse thought thenanopore, the write times W1 (Add “1”),W0 (Add “0”) may need to beadjusted to ensure there is adequate time to perform the desired write(or add) reaction in the Add chamber. Accordingly, the read/writecontroller 6802 (FIG. 68) discuss herein above, may have logic thatmeasures and adjusts for these conditions in real time, for any of theconfigurations and embodiments of fluidic, electrode, or otherconfigurations described herein or others. Also, the traverse time willdepend on the number of bases, the more bases the longer the time. Forexample, for 100K bases, traveling through the nanopore at 1 millionbases per second (typical average velocity for DNA through an nanopore),would take about 100 milliseconds to traverse the nanopore. There mayalso be a delay for entry into the pore, e.g., about 100 milliseconds,however other values may be used.

In addition, during the traverse times T1-T4 shown on the graph 6914,while the memory string (or DNA or polymer) is traversing through thenanopore the read/write controller 6802 may read (or sequence) thevalues of the bits as the pass through the nanopore, as discussedherein. Thus, for each write cycle (Add “1” cycle or Add “0” cycle),there are two time periods T1,T2 or T3,T4, respectively, when the systemmay read the data stored on the memory string. Reading the data on acontinuous basis may be useful for verifying the data, providingmultiple reads of the data, flagging errors in the data, and otherreasons.

There are numerous possible approaches and factors to consider regardingreading the data from the memory string for the present disclosure,including timing (e.g., when and how often to read), fluidic & electrodeand other related configurations (e.g., how to provide the steeringsignals to perform the read), and other factors, which may be determinedbased on the disclosure herein and the design, functional andperformance requirements of the overall memory system.

In some embodiments, the memory string (or DNA or polymer) may only beread when no writing is occurring and the Add chambers have been rinsedand removed of chemical “Add” capability (e.g., addition enzymes, andthe like). In that case, the memory string may be steered into and outof the desired nanopore(s) by the read controller and the informationstored by the read controller for later use. In that case, the readcontroller may communicate with another memory storage device forholding the retrieved data for later use.

The traverse (or transport) times T1,T2,T3,T4 it takes the memory string(or DNA or polymer) to traverse the nanopore (into or out of an Addchamber) may vary based on the length of the memory string (the morebits on the string the longer it will take) and the string transportvelocity through the nanopore (the slower the string moves through thenanopore, the longer it will take). The transport velocity may varybased on a number of factors, including, the angle of the stringapproaching the nanopore, the geometry of the nanopore (cone, cylinder,etc.), the diameter of the nanopore compared to the diameter of thestring (which may vary along its length), the amount of tangling orwrapping or coils in the string, how the velocity varies along thelength of the string, fluid dynamic effects, friction/attraction/bindingwith walls of chamber, viscosity effects, acoustic waves in the fluid,and other factors.

If the velocity is not know accurately, the system may not be able toaccurately determine the number of bits in a word with a long string ofthe same bit state, such as 000000 or 1111111. However, the velocity maybe determined or calibrated by the systems or methods of presentdisclosure by writing a predetermined “velocity calibration sequence” ofdata on the memory string (or DNA or polymer) for a cell, such,alternating 1s and 0s, i.e., 101010101010, and placing it in the storeddata on the string in a known or determinable location, such as near thebeginning of the string or after a certain number of words are written,or after detecting a “special” bit having special properties, such asbeing extra large, as discussed more hereafter. When the system readsthe alternating “1010” pattern, it can determine the velocity of thestring because it knows the pattern. Such a velocity calibrationsequence may be placed a numerous locations along the memory string toenable multiple realtime calibration of the velocity, if desired.

In some embodiments, it may not be necessary to calibrate the velocityif there is a “baseline resolution” between bits, i.e., if the bitsignal goes back to a baseline value prior to the next bit. However, ifthere is a nanopore with a length equal to or longer than a bit, thenbaseline resolution would not be expected. In that case, the systemwould be reading several bits simultaneously and assessing how thatchanges over time, e.g., 110011 to 100111 to 001110, and the like, forthe sequence 1101110. To interpret this scenario effectively, having asmany measurements per unit time as possible is desired. Also, the systemmay perform multiple reads of the same DNA to average out at least someof the variability of the time domain, much of which is random.

The sample voltage values for the steering voltages V_(ST) shown in FIG.69 are for a memory string that has a net (or overall or average)negative charge, such a negatively charged polymer, such as DNA, orother negatively charged polymer. If the memory string has a netpositive charge, the values shown here would be reversed. Other values(and polarities (+/−)) for the memory string (or DNA or polymer)steering voltages shown herein may be used if desired based on theelectronic circuits components and other factors, provided the relativevoltage differences are sufficient to achieve the desire results. Also,it is not necessary that the steering voltages V_(ST) have negative andpositive values. It is only necessary that the relative voltagedifference created by the steering voltages are such that they createthe necessary electric field force to move the memory string through thenanopore 6528 to the desired chamber.

Referring to FIG. 70, a series of time graphs 7000 is shown, having awrite cycle graph 7002 and a corresponding set of bit time graphs7004-70012 showing how 5-bits words would populate for a correspondingfive different bit patterns. In particular, the write (or add) cyclegraph, shows a square wave 7002 indicative of an alternating repeatingwrite cycle of Add “0” cycle, Add “1” cycle, Add “0” cycle, and so on.The time graphs 7004-7012 show an example of five, 5-bit binary datawords 7020 on the left (11100, 00011, 01010, 1111, 0000), andcorresponding time graphs 7004-7012 showing when each bit of the 5-bitdata words 7020 would be written in a single cell using the alternatingwrite cycle 7002 (Add “1”, Add “0”) approach. Cells with an “X” indicateno data is written during that portion of the write cycle 7002. Thegraph also show when each of the data words 7020 would be completelywritten into a cell, shown by an arrow 7014. For Data 11100, it took 9cycles to write, data 00011 took 8 cycles to write, data 01010 took 5cycles to write, 1111 took 10 cycles to write, and data 0000 took 9cycles to write. Thus, number of write cycles (or the time) to write thesame number of bits can vary based on the pattern of the 1's and 0's inthe word, if writing each word to a given cell. In this example, thenumber of write cycles varied from 5 cycles to 10 cycles (i.e., from thenumber of bits to twice the number of bits).

However, if the cells are written (or added) in parallel, i.e., each bitis assigned to a different cell and written simultaneously, the maximumnumber of write cycles would be 2, and the minimum number would be 1,independent of the number of bits or the pattern of the bits. Thus, ifwriting speed is important and an embodiment is used that hasalternating write cycles, formatting the data to be written intoparallel cells instead of writing data words in series to a single cellmay be advantageous. Thus, for some embodiments, there may be atrade-off between write cycle management and data memory cell format.

Referring to FIG. 70A, a flowchart 7030 is shown for the Write/Vst CNTRLLogic 6804 of the Read/Write Memory Controller 6802 (FIG. 68) forwriting bits in accordance with embodiments of the present disclosure.The process/logic 7030 begins at a block 7032, which checks if the writecycle is an Add “0” cycle. If not, the process goes to a block 7034which checks if the write cycle is an Add “0” cycle. If not, the processexits. If the result of block 7034 is YES, a block 7036 sets thesteering voltages V_(ST) to values shown in FIG. 69, e.g., V_(ST1)=GND;V_(ST0)=Neg. Next, a block 7038 checks if the next bit of data to bewritten is a “1”. If NO, the process exits. If YES, a block 7040 setsV_(STDB)=Neg., to release the memory string (or DNA or polymer) into theAdd “1” chamber, for a time t=T1+W1, as shown in FIG. 69. Then, afterthis time has passed, the logic sets V_(STDB)=Pos., to pull the memorystring out of the Add chamber into the Deblock chamber, and the processexits.

If the result of block 7032 is YES, the write cycle is in an Add “0”cycle, and a block 7042 sets V_(ST) to values shown in FIG. 69, e.g.,V_(ST1)=Neg.; V_(ST0)=GND. Next, a block 7044 checks if the next bit ofdata to be written is a “0”. If NO, the process exits. If YES, a block7046 sets V_(STDB)=Neg., to release the memory string (or DNA orpolymer) into the Add “0” chamber, for a time t=T3+W0, as shown in FIG.69. Then, after this time has passed, the logic sets V_(STDB)=Pos., topull the memory string out of the Add chamber into the Deblock chamber,and the process exits. The process 7030 repeats itself continuously tolook for the next write cycle and respond accordingly.

Referring to FIG. 70B, a table showing steps to write “1” and “0” forthe nanopore memory device cell configuration shown in FIG. 66, inparticular, a cell having a common electrode on the bottom for thedeblock chamber and individually controllable electrodes on the top addchambers. There are four steps shown in column 7082 for each type ofwriting and a corresponding controller action shown in column 7084 forthe write controller, and a corresponding result shown in column 7076explaining what happens inside the chip for that particular step.

Referring to FIG. 71, the format of how data is stored may vary based onvarious factors and design criteria. In particular, the memory string(or DNA or polymer) 6550 may be shown as a line 7102 on which are aseries of ovals, indicative of individual “bits” written (or added) onthe memory string 6550 in a given memory cell. In some embodiments, thebits 7104 may be written one after the other to build a “storage word”.A first example data format 7110 shows three components to the storageword 7112, an address section 7106, a data section 7108, and an errorchecking section 7110. The address section 7106 is a label or pointerused by the memory system to locate the desired data. Unlike traditionalsemiconductor memory storage where hardware address lines on a computermemory bus would address a unique memory location, the memory chip andsystem of the present disclosure require the address (or label) to bepart of the data stored and indicative of where the data desired to beretrieved is located. In the examples shown in FIG. 71, the address islocated proximate to or contiguous with the data, as well as errorchecking data, such as parity, checksum, error correction code (ECC),cyclic redundancy check (CRC), or any other form of error checkingand/or security information, including encryption information. In thestorage word 7112, each of the components Address 7106, Data 7108, ErrorChecking 7110, are located after each other in the memory string. Aseach of the components have a known length (number of bits), e.g.,address=32 bits, data=16 bits, error check=8 bits, each storage word7112 and its components can be determined by counting the number ofbits.

Another example data format 7120 shows the same three components,address section 7106, data section 7108, and error checking section7110. However, in between each of the sections there is a “specialbit(s) or sequence” sections S1,S2,S3, shown as numerals 7122,7124,7126,respectively. These special bits S1,S2,S3 may be a predetermined seriesof bits or code that indicate what section is coming next, e.g.,1001001001 may indicate the address is coming next, where as 10101010may indicated the data is coming next, and 1100110011 may indicate theerror checking section in next. In some embodiments, the special bitsmay be a different molecular bit or bit structure attached to thestring, such as dumbbell, flower, or other “large” molecular structurethat is easily definable when it passes through the nanopore. Instead ofit being large it may have other molecular properties that provide aunique change the capacitance or resonance different from the 1 bits and0 bits, as discussed herein above.

Another example data format 7130 shows only Data components 7140 with noaddress component, and an error checking component 7110. In thisstructure, the string holds only the “Data” components and no Addresscomponents, which may be stored in other strings, as discussedhereafter. In this example there are also Special bits S1,S2,S3, shownas numerals 7132,7134,7136, respectively. Similar to the example 7120,these special bits S1,S2,S3 may be a predetermined series of bits orcode that indicate the separation between data sections and indicatewhen an error checking section is next, or may be a different molecularbit or bit structure attached to the string that is easily definablewhen it passes through the nanopore, as discussed herein above.

Referring to FIG. 72, a single row of memory cells 7202-7208 is shown,with an sample memory string 7210-7216, respectively, associated witheach cell. The memory system of the present disclosure is significantlydifferent from traditional semiconductor memory because instead of eachmemory cell storing a single bit of information (1 or 0), each memorycell of the present disclosure can store a significant amount of data.Thus, if the traditional semiconductor memory is viewed as a 2D array,the present memory system is 3D array, where each memory cell locationin the array has significant storage depth. This provides a large rangeof options for how to store data and retrieve data.

For the example shown in FIG. 72, each cell may store a linear selfcontained string of information (storage word), similar to thatdiscussed in the example 7110 of FIG. 71. In that case, each storageword is stored back-to-back on top of other storage words. And each ofthe cells 7202-7208 in the row replicates this structure, and this isrepeated for multiple rows (not shown).

Referring to FIG. 73, in some embodiments, some cells may store onlyaddress information, and some cells only data information. In that case,each row may have a cell, e.g., Cell 1, 7310, which has a memory string7302 of addresses or pointers, and the remainder of the row, e.g.,Cell2-CellN, 7310-7316, respectively, have a corresponding string ofdata 7304-7308, respectively. In that case, the addresses or pointerswould have a value indicative of where the data is stored on the memorychip, such as a row, column and entry number, e.g. Row 3, Column 8,Entry 50, meaning the data corresponding to this address resides at the50^(th) data block in Row 3 and Column 8. This effectively decouples theaddress from being located physically next to the data, which canprovide flexibility in storage. Also, each of the strings may have oneor more error checking or security components to validate theinformation stored on the string. This may be repeated for each row inthe array.

Referring to FIG. 74, instead of storing information contiguously (orserially) on a given memory string, the data may be stored in the memorycell array in parallel. For example, when a storage word is stored, itmay be able to be stored more quickly in a single storage action,storing it across the array, similar to the way traditionalsemiconductor memory works, but allowing it to do it over and over againdue to the 3D depth, each time “pushing” (storing) another storage wordonto the strings. Such a format also enables quick parallel retrieval ofa given storage word (once located). In that case, certain cells 7402may be allocated to storing addresses/pointers, certain cells 7204 maybe allocated to storing parallel data, and certain cells 7406 may beallocated to storing error checking and security data. For example, thestorage word 7210 shown in FIG. 72 (which is stored in series on onestring), may be stored as shown as storage word 7410, having Address1,Data1, and Error Check1, and which is stored in parallel across aplurality of cells (1−N, N+1 to M, and M+1 to P). Similarly, for storageword 7412 which would be stacked across the same strings in parallelwith the storage word 7410 (either underneath or on top of, depending onthe direction of storage on the string). In some embodiments, the datamay be stored in parallel in 2 Dimensions, thereby creating a layered 2Darray of stored information, such as a multi-layered 2D image capturedata may be stored, except allowing a 2D image it to be stored inrealtime, with each 2D snapshot layering on top the prior snap shot.

The bits may be binary bits; however, they are not limited to any basenumbering system as the present disclosure allows the memory stick towrite (or add) more than two different values, as described herein. Inthat case, the cell design would be adjusted accordingly. For examplefor a base-4 system (e.g., GCAT, for DNA based system), there would be 4add chambers and a single de-block chamber, as described herein. Thiscan be extended for any base number system greater than 2, such as 3, 4,5, 6, 7, 8, 9, 10 (decimal), or more, up to N. Where there would be Nadd chambers and 1 deblock chamber. The only limitation would be thatthe chambers are oriented such that the memory string (or DNA orpolymer) can reach all the add chambers.

The term “data” as used herein includes all forms of data including datarepresenting addresses (or labels or pointers, including physical orvirtual), machine code of any type (including but not limited to objectcode, executable code and the like), error checking, encryption,libraries, databases, stacks, and the like that may be stored in memory.In certain examples, such as in FIGS. 71-74 (or elsewhere as the contextimplies), the term “Data” may be shown or described as being separatefrom the “Address,” or “Error Checking”. In those cases, these terms maybe used to show different forms of data for illustrative purposes only.

Chip Fluidics Instrument & Control:

Referring to FIG. 75, the nanopore chip 6700 (FIG. 67) may interact withthe read/write memory controller 6802, as discussed herein above withFIG. 68, which may control the voltages (AC and DC) to steer or controlthe polymer to Add bits and or read the bits on the memory string asshown collectively by lines 7504. The memory chip may also interfacewith an instrument 7502 on lines 7506, which may provide fluidics to thememory chip, such as filling the chip with buffers, enzymes, and/orpolymers or DNA (or other memory strings), as discussed herein. TheInstrument 7502 and the Memory Controller 6802 may be controlled orreceive commands from a Computer System 6870, such at that described andshown with FIG. 68A, that may interact with the user 6878 and may havethe display 6880. The computer system 6870 may interact with theRead/Write Memory Controller 6802 and the Instrument 7502 via thecomputer bus 6872 (FIG. 68). The instrument 7502 has the necessaryelectronics, computer processing power, interfaces, memory, hardware,software, firmware, logic/state machines, databases, microprocessors,communication links, displays or other visual or audio user interfaces,printing devices, and any other input/output interfaces, includingsufficient fluidic and/or pneumatic control, supply and measurementcapability to provide the functions or achieve the results describedherein.

In particular, the instrument may perform the following fluidic actionswith the memory chip: initially fill the chip with the necessary fluids,enzymes, reagents, DNA, or the like through capillary action & or micropumping. For the embodiments where the Add1 and Add0 have flow-throughchannels and deblock as isolated chambers, the deblock chambers could befilled en-masse (via capillary action) first, then sealed—water andbuffers would travel into the add chambers which could then be filledwith their enzymes/buffers OR deblock chambers could be individuallyfilled via targeted addition (e.g., ink-jet) and dried and sealed. Inthat case, the Add chambers may be filled under vacuum to ensure nobubbles get trapped in the deblock chamber, or the deblock chambers maybe sealed with a material which allows gas but not water to pass through(such as PDMS). Also, the deblock chambers may be filled by leaving thebottom of the cell open during assembly, and placing the cell bottom inthe desired fluid, and the fluid will wick up into the deblock chambersby capillary action.

There are various fluidics designs that will achieve the desired resultsfor fluidic filling and flushing. For example, the Add “0” channels andAdd “1” channels may respectively be connected together (like channelstogether) in a continuous serpentine (back and forth) pattern, and fedfluid through vias from a layer above the channels. The vias may connectto the instrument via standard fluidic interfaces sufficient to supplythe desired fluids to the channels. In some embodiments, the Addchannels may each be fed through separate vias from a common reservoirfor Add “0” channels and from a separate common reservoir for Add “1”channels located on a layer above the channels. Any other fluidic designmay be used if desired. Sample dimensions for the Add channels, are:width about 100 nm to about 10 microns, height of about 1 micron toabout 50 microns, and length of about 100 mm (1 cm or 1000 microns) fromone side of the chip to the other. A serpentine connected channel wouldbe a multiple of this depending on how many channels are connected inseries.

The instrument 7502 may also be used during initialization and celltesting if desired. For example, for cell initialization & cell testingQuality Control (QC) for nanopore quality to ensure expected current isobserved (current proportional to pore size). Also, QC for DNA presence:ensure that the expected current (or capacitance or impedance, or shiftin magnitude or phase of the resonance, as discussed herein) changescharacteristic of DNA (or polymer, etc.) moving though the nanopore(e.g., expected reduction in current, or shift in magnitude or phase ofthe resonance, as discussed herein). In addition, it may be used for QCfor circuit formation which would be similar to that performed fornanopore quality.

The instrument 7502 may also be used for DNA addition, as previouslydescribed herein, where DNA with origami is introduced via one of theadd chambers (or channels), current may be applied to cells untilinsertion is detected, modified DNA end in deblock chamber diffuses andthen attaches to surface, and restriction enzyme introduced to addchamber to cleave origami which is then removed via buffer flow.

In another embodiment, the invention provides a single or doublestranded DNA molecule as described above, wherein the single strand orthe coding sequence consists essentially of nonhybridizing bases, forexample adenines and cytosines (As and Cs), which are arranged insequence to correspond to a binary code, e.g., for use in a method ofdata storage. For example, the invention provides DNA (DNA 1), whereinthe DNA is single or double stranded, at least 1000 nucleotides long,e.g., 1000-1,000,000 nucleotides or, for example, 5,000 to 20,000nucleotides long, wherein the sequence of the nucleotides corresponds toa binary code; e.g.,

-   -   1.1. DNA 1 wherein the DNA is single stranded.    -   1.2. DNA 1 wherein the DNA is double stranded.    -   1.3. Any foregoing DNA wherein the nucleotides in a single        strand or in the coding strand are selected from adenine,        thymine and cytosine nucleotides, e.g. are selected from adenine        and cytosine nucleotides or thymine and cytosine nucleotides    -   1.4. Any foregoing DNA consisting primarily of nonhybridizing        nucleotides, so that it will not form significant secondary        structures when in the form of a single strand.    -   1.5. Any foregoing DNA wherein the nucleotides are at least 95%,        e.g. 99%, e.g., 100% adenine and cytosine nucleotides.    -   1.6. Any foregoing DNA comprising a nucleotide or sequence of        nucleotides added to separate or punctuate the nucleotides        comprising a binary code, e.g., to separate the 1's and 0's or        groups of 1's and 0's, so that consecutive 1's or 0's can be        more easily read.    -   1.7. Any foregoing DNA wherein (a) each bit in the binary code        corresponds to a single nucleotide, e.g. each of 1 and 0        correspond to A or C; or (b) each bit in the binary code        corresponds to a series of more than 1 nucleotides, e.g. 2, 3 or        4 nucleotides, e.g., AAA or CCC.    -   1.8. Any foregoing DNA which is crystallized.    -   1.9. Any foregoing DNA which is provided in a dry form together        with one or more of a buffer salt (e.g., a borate buffer), an        antioxidant, a humectant, e.g. a polyol, and optionally a        chelator, for example as described in U.S. Pat. No. 8,283,165        B2, incorporated herein by reference; and/or in a matrix between        the nucleic acid and a polymer, such as poly(ethylene        glycol)-poly(l-lysine) (PEG-PLL) AB type block copolymer; and/or        together with a complementary nucleic acid strand or a protein        that binds the DNA.    -   1.10. Any foregoing DNA which contains an identifying sequence.    -   1.11. Any foregoing DNA that contains PCR amplification        sequences.    -   1.12. Any foregoing DNA that contains one or more calibrating        sequences, e.g., known sequences of nucleotides which can be        used to calibrate a nanopore-based sequencing device, e.g. to        measure the speed of the DNA passage through the nanopore or the        relative effect on capacitance or current attributable to        different nucleotides passing through the nanopore.    -   1.13. Any foregoing DNA which contains a terminal linker group        enabling it to be anchored to a surface near the nanopore in a        nanopore-based device such as Nanochip 1, et seq; a spacer        sequence long enough to permit the DNA strand to reach the        nanopore when anchored to a surface, a data storage sequence        wherein the sequence encodes data, codons or other information,        and optionally a restriction sequence, enabling the DNA to be        cleaved and retrieved once synthesized.    -   1.14. Any foregoing DNA made by any of Method 1 et seq. or        Method 2 et seq. or Method A, et seq.

In yet another embodiment, the invention provides the use of any of DNA1, et seq. in a method for storing information.

In another embodiment, the invention provides the use of a singlestranded DNA in a method for storing information, e.g., wherein thesequence is substantially non-self-hybridizing.

The nanochips can be fabricated for example as depicted in FIGS. 23-29.For example, in one format, each polymer strand is associated with twoor four addition chambers, wherein the two addition chamber format isuseful for encoding binary code in the polymer, and the four additionchamber format is particularly useful for making custom DNA sequences.Each addition chamber contains a separately controllable electrode. Theaddition chambers contain reagents to add monomers to the polymer inbuffer. The addition chambers are separated by a membrane comprising oneor more nanopores from a reserve chamber, which may be common tomultiple addition chambers, and which contains deprotection reagents andbuffer, to deprotect the protected monomers or oligomers added in theaddition chambers. The nanochips comprise a multiplicity of additionchamber sets, to allow parallel synthesis of many polymers.

For example, in some embodiments, the nanopore-based memory device ofthe present disclosure may be fabricated on a polished single-crystalsilicon wafer, e.g., approximately 400 microns thick. A layer of siliconnitride of about 200 nm thick is deposited onto both sides of thesilicon wafer via, e.g., low pressure chemical vapor deposition, orother similar technique. Next, a layer of silicon dioxide, e.g., about 5micron thick, is deposited on the top side of the single-crystal siliconwafer and then polished. Next, on top of the Silicon dioxide layer, athin (e.g., about 5 nm) layer of silicon nitride is deposited. Then, alayer of silicon dioxide (e.g., about 5 micron) is deposited. On top ofthe silicon dioxide layer, a layer of silicon nitride, e.g., about ˜200nm thick, is deposited. Next, the fluidic “Add” Channels are created byetching through the top layer of silicon nitride and through the silicondioxide, exposing the thin silicon nitride layer at the bottom of thechannels. These channels will become the ‘Add 1’ and ‘Add 0’ channels.Another silicon wafer (approximately 300 microns thick, with insulated(using, for example, silicon dioxide) vias filled with conductive metalto serve as wiring), is aligned to the original silicon wafer and thewafers bonded together. The bonded wafer is then etched from the bottomof the device through the silicon nitride, then silicon layer. Afterthis, the individual deblock chambers are etched in the silicon dioxide,exposing the thin silicon nitride layer. Nanopores are then created inappropriate locations in the thin silicon nitride layer using electronbeams or other appropriate techniques. An additional silicon wafer(approximately 300 microns thick, with insulated (via, for example,silicon dioxide) vias filled with conductive metal to serve as wiring)is aligned to and bonded to the bottom of the bonded wafer. Fluidicinlets and outlets are introduced by etching or drilling into the toplayer of the device, through to the fluidic channels. Connections to theelectrodes embedded within the device (which connect internally to thefluidic channels and deblock chambers), can be accessed on the top anbottom surfaces of the device.

High-bandwidth and low-noise nanopore sensor and detection electronicsare important to achieving single-DNA base resolution. In certainembodiments, the nanochip is electrically linked to a ComplementaryMetal-Oxide Semiconductor (CMOS) chip. Solid-state nanopores can beintegrated within a CMOS platform, in close proximity to the biasingelectrodes and custom-designed amplifier electronics, e.g., as describedin Uddin, et al., “Integration of solid-state nanopores in a 0.5 μm cmosfoundry process”, Nanotechnology (2013) 24(15): 155501, the contents ofwhich are incorporated herein by reference.

In another embodiment, the disclosure provides a nanochip (Nanochip 1)for synthesis of and/or sequencing an electrically charged polymer,e.g., DNA, comprising at least two distinct monomers, the nanochipcomprising at least a first and second reaction chambers, separated by amembrane comprising one or more nanopores, wherein each reaction chambercomprises one or more electrodes to draw the electrically chargedpolymer into the chamber and further comprises an electrolytic media andoptionally reagents for addition of monomers to the polymer, forexample,

-   -   1.1. Nanochip 1 wherein the nanopore has a diameter of 2-20 nm,        e.g. 2-10 nm, for example 2-5 nm.    -   1.2. Any foregoing nanochip wherein the some or all of the walls        of the reaction chambers of the nanochip comprise a silicon        material, e.g., silicon, silicon dioxide, silicon nitride, or        combinations thereof, for example silicon nitride.    -   1.3. Any foregoing nanochip wherein the some or all of the walls        of the reaction chambers of the nanochip comprise a silicon        material, e.g., silicon, silicon dioxide, silicon nitride, or        combinations thereof, for example silicon nitride, and some or        all of the nanopores are made by ion bombardment.    -   1.4. Any foregoing nanochip wherein some or all of the nanopores        are comprised of a pore-forming protein, α-hemolysin, in a        membrane, e.g. a lipid bilayer.    -   1.5. Any foregoing nanochip wherein some or all of the walls of        the reaction chambers are coated to minimize interactions with        the reagents, e.g., coated with a polymer such as polyethylene        glycol, or with a protein, such a bovine serum albumin.    -   1.6. Any foregoing nanochip comprising an electrolyte media.    -   1.7. Any foregoing nanochip comprising an electrolyte media        comprising a buffer, e.g., a buffer for pH 7-8.5, e.g. ca. pH 8,        e,g, a buffer comprising tris(hydroxymethyl)aminomethane (Tris),        a suitable acid, and optionally a chelater, e.g.,        ethylenediaminetetraacetic acid (EDTA), for example TAE buffer        containing a mixture of Tris base, acetic acid and EDTA or TBE        buffer comprising a mixture of Tris base, boric acid and EDTA;        for example a solution comprising 10 mM Tris pH 8, 1 mM EDTA,        150 mM KCl, or for example, 50 mM Potassium Acetate, 20 mM        Tris-acetate, 10 mM Magnesium Acetate, pH 7.9 @ 25° C.    -   1.8. Any foregoing nanochip comprising reagents for addition of        monomers to the polymer.    -   1.9. Any foregoing nanochip capable of both synthesizing        (“writing”, e.g., by adding monomers or groups of monomers        sequentially to the polymer) and sequencing (“reading”, e.g., by        measuring changes in current and/or inductance as the monomers        pass through the nanopore) the polymer.    -   1.10. Any foregoing nanochip wherein the membrane comprising one        or more nanopores comprises a metal surface on both sides, the        metal surface being separated by an insulator, e.g. a silicon        nitride membrane, the metal surfaces being configured, e.g., by        lithographic means, to provide electrodes at either end of each        nanopore, e.g., such that a current flow across the nanopore may        be established through the nanopore via an electrolyte media,        e.g., such that the currant can draw the polymer through the        nanopore and as the polymer passes through the nanopore, the        change in electric potential across the nanopore can be measured        and used to identify the sequence of monomers in the polymer.    -   1.11. Any foregoing nanochip comprising an electrically charged        polymer which is DNA.    -   1.12. Any foregoing nanochip comprising an electrically charged        polymer which is single stranded DNA (ssDNA).    -   1.13. Any foregoing nanochip comprising an electrically charged        polymer which is DNA comprising a predetermined restriction        site.    -   1.14. Any foregoing nanochip comprising an electrically charged        polymer which is DNA wherein the DNA is a DNA as described in        any of DNA 1, et seq., above.    -   1.15. Any foregoing nanochip comprising an electrically charged        polymer which is DNA, wherein the DNA comprises at least 95%,        e.g. 99%, e.g., 100% adenines and cytosines.    -   1.16. Any foregoing nanochip comprising an electrically charged        polymer which is DNA, wherein the DNA comprises only adenines        and cytosines.    -   1.17. Any foregoing nanochip comprising one or more ports to        permit introduction of and flushing out of buffer and reagents.    -   1.18. Any foregoing nanochip comprising a buffer solution, e.g.,        a solution comprising a buffer for pH 7-8.5, e.g. ca. pH 8, e,g,        a buffer comprising tris(hydroxymethyl)aminomethane (Tris), a        suitable acid, and optionally a chelater, e.g.,        ethylenediaminetetraacetic acid (EDTA), for example TAE buffer        containing a mixture of Tris base, acetic acid and EDTA or TBE        buffer comprising a mixture of Tris base, boric acid and EDTA;        for example a solution comprising 10 mM Tris pH 8, 1 mM EDTA,        150 mM KCl, or for example, 50 mM Potassium Acetate, 20 mM        Tris-acetate, 10 mM Magnesium Acetate, pH 7.9 @ 25° C.    -   1.19. Any foregoing nanochip which is or is capable of being        lyophilized for storage and subsequently rehydrated, e.g.,        wherein the structure of the nanochip comprises a hydratable or        water permeable polymer.    -   1.20. Any foregoing nanochip which is synthesized in a dry form,        e.g., wherein the structure of the nanochip comprises a        hydratable or water permeable polymer, followed by hydration        prior to use, optionally followed by lyophilization for long        term storage once the write process is complete.    -   1.21. Any foregoing nanochip wherein the electrically charged        polymer, e.g., DNA, is stabilized with histone.    -   1.22. Any foregoing nanochip wherein the interior surface is        positively charged.    -   1.23. Any foregoing nanochip wherein the electrodes are operably        connected in a capacitive circuit capable of providing a        radiofrequency pulsating direct current, e.g. at a frequency of        1 MHz to 1 GHz, e.g. 50-200 MHz, for example about 100 MHz,        across the nanopore, e.g., wherein the pulsating direct current        can draw the charged polymer through the nanopore and the        monomer sequence can be determined by measuring the capacitive        variance across the nanopore as the charged polymer goes through        the nanopore.    -   1.24. Any foregoing nanochip comprising a reserve or deblocker        chamber, which contains reagents for deprotection of the polymer        following addition of a monomer or oligomer in one of addition        chambers.    -   1.25. Any foregoing nanochip comprising a multiplicity of pairs        of addition chambers.    -   1.26. Any foregoing nanochip comprising an electrical control        layer, a fluidics layer and an electrical ground layer, e.g., as        depicted in FIG. 24, joined by wafer bonding.    -   1.27. Any foregoing nanochip wherein the nanopore is made by        drilling with FIB, TEM, wet or dry etching.    -   1.28. Any foregoing nanochip wherein the membrane comprising the        nanopores is from 1 atomic layer to 30 nm thick.    -   1.29. Any foregoing nanochip wherein the membrane comprising the        nanopores is made of SiN, BN, SiOx, Graphene, transition metal        dichalcogenides e.g. WS₂ or MoS₂.    -   1.30. Any foregoing nanochip comprising wiring made from metal        or polysilicon.    -   1.31. Any foregoing nanochip wherein the wiring density is        increased by 3D stacking, with electrical isolation provided by        dielectric deposition (e.g., via PECVD, sputtering, ALD etc).    -   1.32. Any foregoing nanochip wherein the contact to the        electrode in the addition chamber is made using Through Silicon        Via (TSV) by Deep Reactive Ion Etch (DRIE), e.g. using cryo or        BOSCH process, or via wet silicon etching.    -   1.33. Any foregoing nanochip wherein individual voltage control        for the electrode in each addition chamber allows the electrode        in each addition chamber to be controlled and monitored        individually.    -   1.34. Any foregoing nanochip wherein each polymer is associated        with a first addition chamber, a second addition chamber, and a        deblocking chamber.    -   1.35. Any foregoing nanochip wherein one or more chambers have        fluid flow.    -   1.36. Any foregoing nanochip wherein one or more chambers are        fluidically isolated.    -   1.37. Any foregoing nanochip wherein the deblocking chamber has        fluid flow.    -   1.38. Any foregoing nanochip wherein addition chambers have        common fluid flow.    -   1.39. Any foregoing nanochip wherein wiring between chambers is        common among chambers of similar type (e.g. among first addition        chambers, among second addition chambers, and among deblocking        chambers.)    -   1.40. Any foregoing nanochip wherein the addition chambers have        individual voltage control and the deblocking chambers have a        common electrical ground.    -   1.41. Any foregoing nanochip wherein the deblocking chambers        have individual voltage control, the first addition chambers        have a common electrical ground and the second addition chambers        have a common electrical ground.    -   1.42. Any foregoing nanochip wherein the nanochip is fabricated        by wafer bonding, and the chambers are prefilled with desired        reagents prior to bonding.    -   1.43. Any foregoing nanochip wherein one or more internal        surfaces are silanized.    -   1.44. Any foregoing nanochip which has one or more ports for        introduction or removal of fluid.    -   1.45. Any foregoing nanochip wherein the electrodes in the        chambers are restricted from direct contact with the charged        polymer, e.g., wherein the electrode is placed too far from the        nanopore to be reached by a charged polymer bound to a surface        adjacent to the nanopore, or wherein the electrode is protected        by a material which will permit the passage of water and single        atom ions (e.g., Na+, K+ and Cl− ions) but not the passage of        the polymer or monomer or oligomer reagents to be joined to the        polymer.    -   1.46. Any foregoing nanochip which is electrically linked to a        Complementary Metal-Oxide Semiconductor (CMOS) chip.    -   1.47. Any foregoing nanochip which is operably linked to a chip        controller, as hereinbefore described.

For example, in one embodiment, the invention provides a nanochip, e.g.,according to any of Nanochip 1, et seq., for sequencing an electricallycharged polymer, e.g., DNA, comprising at least two distinct monomers,the nanochip comprising at least a first and second reaction chamberscomprising an electrolyte media and separated by a membrane comprisingone or more nanopores, wherein each reaction chamber comprises at leastone pair of electrodes disposed on opposite sides of the membrane,wherein the electrodes are operably connected in a capacitive circuitcapable of providing a radiofrequency pulsating direct current, e.g. ata frequency of 1 MHz to 1 GHz, e.g. 50-200 MHz, for example about 100MHz, across the nanopore, e.g., wherein the pulsating direct current candraw the charged polymer through the nanopore and the monomer sequencecan be determined by measuring the capacitive variance across thenanopore as the charged polymer goes through the nanopore.

In another embodiment, the invention provides a method of reading amonomer sequence of a charged polymer comprising at least two differenttypes of monomers, for example a DNA molecule, comprising applying aradiofrequency pulsating direct current, e.g. at a frequency of 1 MHz to1 GHz, e.g. 50-200 MHz, for example about 100 MHz, across a nanopore,wherein the pulsating direct current draws the charged polymer throughthe nanopore and the monomer sequence is read by measuring thecapacitive variance across the nanopore as the charged polymer goesthrough the nanopore, for example wherein the circuit is a resonantcircuit and the capacitive variance is detected by detecting changes inthe resonant frequency.

For example, in a particular embodiment, the invention provides ananopore-based device (Device 1), e.g., a nanochip, e.g., according toany of Nanochip 1, et seq., which is capable of reading data stored in apolymer, the device comprising:

-   -   a. a resonator having an inductor and a cell, the cell having a        nano-pore and a polymer that can traverse through the nanopore,        the resonator having an AC output voltage frequency response at        a probe frequency in response to an AC input voltage at the        probe frequency;    -   b. an AC input voltage source configured to provide an AC input        voltage at least the probe frequency; and    -   c. a monitoring device configured to monitor the AC output        voltage at least at the probe frequency, the AC output voltage        at the probe frequency being indicative of the data stored in        the polymer at the time monitoring.

For example, in certain embodiments of Device 1 the polymer comprises atleast two monomers having different properties causing differentresonant frequency responses at the probe frequency the responseindicative of at least two different data bits, e.g., two different DNAnucleotides or oligonucleotides; and/or the inductor is connected inseries with an effective capacitance to create the resonator, acombination of the inductor and effective capacitance being related tothe resonant frequency response at the probe frequency.

For example, in a particular embodiment, the invention provides a method(Method 3) for reading data stored in a polymer, e.g., in conjunctionwith any of Methods 1, et seq., Method A, et seq., or Method 2, et seq.,e.g., using a device according to any of Nanopore 1, et seq., or Device1, et seq. comprising:

-   -   a. providing a resonator having an inductor and a cell, the cell        having a nanopore and a polymer that can traverse through the        nanopore, the resonator having an AC output voltage frequency        response at a probe frequency in response to an AC input voltage        at the probe frequency;    -   b. providing the AC input voltage having at least the probe        frequency; and    -   c. monitoring the AC output voltage at least at the probe        frequency, the AC output voltage at the probe frequency being        indicative of the data stored in the polymer at the time of        monitoring; for example,

-   3.1. Method 3, wherein the polymer comprises at least two different    types of monomers or oligomers having different properties causing    different resonant frequency responses.

-   3.2. Method 3.1 wherein the at least two different types of monomers    or oligomers comprises at least a first monomer or oligomer having a    first property that causes a first resonant frequency response when    the first monomer or oligomer is in the nanopore, and a second    monomer or oligomer having a second property that causes a second    resonant frequency response when the second monomer or oligomer is    in the nanopore.

-   3.3. Method 3.2, wherein a characteristic of the first frequency    response at the probe frequency is different from the same    characteristic of the second frequency response at the probe    frequency.

-   3.4. Method 3.3 wherein the characteristic of the first and second    frequency responses comprises at least one of magnitude and phase    response.

-   3.5. Method 3.4 wherein the first property and the second property    of the monomers comprises a dielectric property.

-   3.6. Any foregoing method wherein the first and second monomers or    oligomers each comprises a plurality of monomers or oligomers.

-   3.7. Any foregoing method wherein the cell comprises at least a top    and bottom electrode, the nanopore being disposed between the    electrodes, and the cell having a fluid therein, and wherein the    electrodes, the nanopore and the fluid having an effective cell    capacitance that changes when the polymer passes through the    nanopore.

-   3.8. Any foregoing method wherein the inductor is connected in    series with the effective capacitance to create the resonator, a    combination of the inductor and effective capacitance being related    to the resonant frequency response.

-   3.9. Any foregoing method wherein the polymer is moved through the    nanopore via a DC steering voltage applied to the electrodes.

-   3.10. Any foregoing method wherein the cell has at least three    chambers, at least two nanopores, and at least three electrodes for    moving the polymer through the nanopore.

-   3.11. Any foregoing method wherein at least two of the monomers is    indicative of at least two different bits of data.

-   3.12. Any foregoing method wherein a plurality of monomers is    indicative of a bit of data.

-   3.13. Any foregoing method wherein the polymer comprises DNA, and    wherein the DNA comprises bases, at least two of the bases providing    a unique frequency response at the probe frequency.

-   3.14. Any foregoing method wherein the probe frequency is about 1    MHz to 1 GHz.

-   3.15. Any foregoing method wherein at least two of the monomers have    a dielectric property that affects the frequency response of the    resonator to produce at least two different frequency responses at    the probe frequency.

-   3.16. Any foregoing method wherein the polymer comprises DNA and the    sequence encodes a protein or a biologically functional RNA, e.g.,    mRNA.

-   3.17. Any foregoing method wherein the sequence encodes    computer-readable data, e.g., in a binary, ternary or quaternary    code.

-   3.18. Any foregoing method which is a method to read or confirm the    sequence of a polymer sequenced in accordance with any of Methods 1,    et seq., Methods A, et seq, or Methods 2 et seq.

In another embodiment, the invention provides a method for storing andreading data on a polymer in situ in a nanopore-based chip, comprising:

-   -   a. providing a cell having at least three chambers, including an        Add “1” chamber arranged to add a “1” bit to the polymer and an        Add “0” chamber arranged to add a “0” bit to the polymer, and a        “deblock” chamber arranged to enable the polymer to receive the        “1” bit and “0” bit when the polymer enters the Add “1” or Add        “0” chambers, respectively;    -   b. successively steering the polymer from the “deblock” chamber        through the nanopore to the Add “1” chamber or to the Add “0”        chamber based on a predetermined digital data pattern to create        the digital data pattern on the polymer; and    -   c. reading the digital data stored on the polymer as it passes        through the nanopore using a resonance frequency response of a        nanopore-polymer resonator (NPR) on the chip, for example using        a method according to Method 3, et seq.

In another embodiment, the invention provides a method of data storageand device, using a nanochip, e.g., any of Nanochip 1 et seq. to make anelectrically charged polymer, e.g., DNA, comprising at least twodistinct monomers or oligomers, wherein the monomers or oligomers arearranged in sequence to correspond to a binary code, e.g., in accordancewith any of the foregoing Methods 1 and/or 2 et seq.

For example, in one embodiment, the nanochip comprising the polymer thussynthesized provides a data storage device, as the nanochip can beactivated and the sequence of the polymer detected by passing it througha nonopore at any time. In other embodiments the polymer is removed fromthe nanochip, or amplified and the amplified polymer removed from thenanochip, stored until required, and then read using a conventionalsequencer, e.g., a conventional nanopore sequencing device,

In another embodiment, the invention provides a method of storinginformation comprising synthesizing any of DNA 1, et seq., e.g., inaccordance with any of Methods 1, et seq. or Methods 2, et seq.

In another embodiment, the invention provides a method of reading abinary code, e.g., as encoded on any of DNA 1, et seq., using a nanoporesequencer, for example using Nanochip 1 et seq., as described herein.

In another embodiment, the invention provides any foregoing methodwherein the nanochip is erased using an enzyme which lyses the chargedpolymer, e.g., a deoxyribonuclease (DNase) to hydrolyze DNA.

All dimensions described herein are shown for exemplary embodiments ofthe present disclosure, other dimensions, geometries, layouts, andorientations may be used if desired, provided they provide the functionsdescribed herein.

Also, the present disclosure is not limited to use with DNA based datastorage, and may be used with any type of molecular data storage, suchas any polymer or other material that has the necessary properties toachieve the functions or performance described herein.

Any automated or semi-automated device or component described herein maybe a computer-controlled device having the necessary electronics,computer processing power, interfaces, memory, hardware, software,firmware, logic/state machines, databases, microprocessors,communication links, displays or other visual or audio user interfaces,printing devices, and any other input/output interfaces, includingsufficient fluidic and pneumatic control, supply and measurementcapability to provide the functions or achieve the results describedherein. Except as otherwise explicitly or implicitly indicated herein,process or method steps described herein may be implemented withinsoftware modules (or computer programs) executed on one or more generalpurpose computers. Specially designed hardware may alternatively be usedto perform certain operations. In addition, computers or computer-baseddevices described herein may include any number of computing devicescapable of performing the functions described herein, including but notlimited to: tablets, laptop computers, desktop computers and the like.

Although the disclosure has been described herein using exemplarytechniques, algorithms, or processes for implementing the presentdisclosure, it should be understood by those skilled in the art thatother techniques, algorithms and processes or other combinations andsequences of the techniques, algorithms and processes described hereinmay be used or performed that achieve the same function(s) and result(s)described herein and which are included within the scope of the presentdisclosure.

Any process descriptions, steps, or blocks in process flow diagramsprovided herein indicate one potential implementation, and alternateimplementations are included within the scope of the preferredembodiments of the systems and methods described herein in whichfunctions or steps may be deleted or performed out of order from thatshown or discussed, including substantially concurrently or in reverseorder, depending on the functionality involved, as would be understoodby those reasonably skilled in the art.

It should be understood that, unless otherwise explicitly or implicitlyindicated herein, any of the features, characteristics, alternatives ormodifications described regarding a particular embodiment herein mayalso be applied, used, or incorporated with any other embodimentdescribed herein. Also, the drawings herein are not drawn to scale,unless indicated otherwise.

EXAMPLES Example 1—Immobilizing One End of DNA Adjacent to Nanopore andControlled Back and Forth Movement of DNA Via Electrical Current

Experimental procedures are developed to demonstrate that DNA is movedback and forth between two chambers separated by a nanopore, via anelectrical current, under conditions that a relevant protein does notmove between chambers.

A nanochip comprising two chambers is fabricated from silicon nitride.Nanopores of <4 nm (for dsDNA or ssDNA) and 2 nm (for ssDNA only) areprepared as described in Briggs K, et al. Automated fabrication of 2-nmsolid-state nanopores for nucleic acid analysis, Small(2014)10(10):2077-86. The two chambers are referred to as a ‘near’ and‘far’ chamber, the far chamber being the chamber where 3′ end of DNA isconjugated.

It is shown that ssDNA (2 nm pore) and ssDNA+dsDNA (4 nm pore) but notprotein pass through the nanopore. Passing through the nanopores isdetected by electrical current disruption.

Conjugation of DNA to Pore Surface:

Attach 5′ amino modified DNA to carboxy-coated polystyrene beads(Fluoresbrite® BB Carboxylate Microspheres 0.05 m, from Polysciences,Inc.) via carbodiimide mediated attachment. 3′ of DNA is labeled withbiotin. DNA is of a pre-specified length.

Strepatividin Conjugation:

Conjugation was performed on the ‘far’ side of a silicon nitridenanopore conjugate streptavidin to the surface, as described in Arafat,A. Covalent Biofunctionalization of Silicon Nitride Surfaces. Langmuir(2007) 23 (11): 6233-6244.

Immobilization of DNA Near the Nanopore:

DNA conjugated polystyrene beads in buffer is added to ‘near’ chamberand buffer is added to ‘far’ chamber (standard buffer: 10 mM Tris pH 8,1 mM EDTA, 150 mM KCl). Voltage (˜100 mV) is applied until currentdisruption is observed (use an Axon Nanopatch200B patch-clampamplifier). 50 nm beads cannot pass through the nanopore, so when a DNAstrand has gone through and a bead is pressed against an end of thenanopore the current is highly disrupted. Current is maintained 1-2 minsuntil DNA is irreversibly bound to immobilized streptavidin on the farside via binding of biotin. To confirm that the DNA has beenimmobilized, the current is reversed. Different currents are observed ifDNA is in or out of the pore. If it appears that DNA is not immobilized,then the process is repeated.

Release the Bead Via Endonuclease:

Restriction enzyme in restriction enzyme buffer is added to the chamberwhere the DNA is attached. In one embodiment, the DNA is single strandedand contains a restriction site cleavable by an enzyme that will cleavesingle stranded DNA. See, e.g., Nishigaki, K., Type II restrictionendonucleases cleave single-stranded DNAs in general. Nucleic Acids Res.(1985) 13(16): 5747-5760. In an alternative embodiment, a complementaryoligonucleotide is added to the chamber where the DNA is attached andallowed to hybridize for 30 minutes to create dsDNA, then therestriction enzyme is added. Once the bead is released, it is washedaway. Current is switched between forward and reverse to confirm thatthe DNA goes into/through and out of the pore.

Demonstrate Controlled Back and Forth Movement:

Using standard buffer, current is applied in forward direction untilsignal disruption is observed and then reverted to ‘normal’ after theDNA passes. Reverse current is applied until signal disruption isobserved. It is observed that the signal does not go back to normal asthe DNA remains in the pore. Application of current in forward andreverse direction is repeated for several cycles to confirm that DNAmoves back and forth through the nanopore.

Example 1a: Immobilizing DNA Strand Adjacent to Nanopore in a SiliconDioxide Chip

A nanochip interior wall is fabricated from silicon dioxide. Both sidesare silanized, but the oligonucleotide is conjugated to just one side ofthe chip wall, then a nanopore is created.

Silanization:

The surface of the chip wall is cleaned with piranha solution (variousbrands commercially available, generally comprising a mixture ofsulfuric acid (H₂SO₄) and hydrogen peroxide (H₂O₂), which remove organicresidues from the surface) at 30° C., and washed with double-distilledwater. A stock solution of (3-aminopropyl)triethoxysilane (APTES) isprepared, with 50% methanol (MeOH), 47.5% APTES, 2.5% nanopure H2O, andaged >1 hour at 4° C. The APTES stock is then diluted 1:500 in MeOH andapplied to and incubated with the chip wall at room temperature. Thechip wall is then rinsed with MeOH and dried at 110° C. for 30 minutes.

Conjugation:

The chip wall is then incubated for 5 hours at room temperature in a0.5% w/v solution of 1,4-phenylene diisothiocyanate (PDC) in dimethylsulfoxide (DMSO). It is washed briefly twice with DMSO and the brieflytwice with double distilled water. The chip wall is then incubated with100 nM amine-modified single stranded DNA oligomers (ca. 50-mers) indouble distilled water (pH 8) overnight at 37° C. Then the chip wall iswashed twice with 28% ammonia solution to deactivate any unreactedmaterial, and washed twice with double distilled water. One or morenanopores are then created in the wall.

Once the fabrication of the nanochip is complete, the interior wall iscoated with DNA oligomers ca. 50 bp long. This permits a single strandedDNA having an end-terminal sequence complementary to the surface boundDNA to be localized to a nanopore by attaching the ssDNA to a relativelybulky structure (e.g. a bead, a protein, or a DNA origami structurehaving a diameter too large to fit through the nanopore,), wherein thesequence complementary to the surface-bound DNA is distal to the bulkystructure, pulling the charged polymer through the nanopore usingcurrent, allowing the ssDNA to bind to a complementary surface bound DNAoligomer adjacent to the nanopore, and cleaving off the bulky structure.

Example 2: DNA Synthesis—Single Nucleotide Addition

DNA is moved to ‘reserve’ chamber by applying appropriate current anddetecting DNA movement.

Terminal transferase enzyme (TdT, New England Biolabs) in appropriatebuffer (50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM MagnesiumAcetate, pH 7.9 @ 25° C.), plus reversibly blocked-dATP* is added to the‘addition’ chamber. The buffer is also added to the ‘reserve’ chamber.

dNTPs that have reversible blocks on the 3′-OH are used to addnucleotides to the DNA. When added to the DNA chain, the next dNTPcannot be added until the blocked dNTP is unblocked.

Deblocking can be chemical or enzymatic. Different approaches areutilized:

a. 3′ O-allyl: Allyl is removed by Pd-catalyzed deallylation in aqueousbuffer solution as described in Ju J, Four-color DNA sequencing bysynthesis using cleavable fluorescent nucleotide reversible terminators.Proc Natl Acad Sci USA. (2006); 103(52):19635-40; or by using iodine (10mol %) in polyethylene glycol-400, as described in Shankaraiah G., etal., Rapid and selective deallylation of allyl ethers and esters usingiodine in polyethylene glycol-400. Green Chem. (2011)13: 2354-2358

b. 3′ O—NH2: Amine is removed in buffered NaNO₂, as described in U.S.Pat. No. 8,034,923.

c. 3′-phosphate. Phosphate is hydrolyzed with Endonuclease IV (NewEngland Biolabs). Other possible 3′ modifications which can also beremoved with Endonuclease IV include phosphoglycoaldehyde anddeoxyribose-5-phosphate.

d. 3′-O-Ac: Acetate is removed by enzymatic hydrolysis as described inUd-Dean, A theoretical model for template free synthesis of long DNAsequence. Syst Synth Biol (2008) 2:67-73,

The DNA is then moved to the ‘far’ chamber by applying appropriatecurrent and detecting DNA movement. DNA is deprotected by switching outbuffers and adding deblocking buffer/solution as described in a.-d.above.

Process is repeated as desired to make sequence of interest.

Example 3: DNA Synthesis: Block Oligonucleotide Addition

The 3′ end of double stranded DNA is attached adjacent to a nanoporewith 4 nm aperture. The 5′ end of the DNA has an overhang of CG (readingfrom 5′ to 3′).

Oligo cassettes A and B are made as follows:

(SEQ ID NO 1) 5′ CGAAGGG <CODE A OR B> GTCGACNNNNN 3′ GCTTCCC<COMPLEMENT> CAGCTGNNNNN

CodeA and CodeB each represent an informational sequence. Ns refer toany nucleotide. The 5′ sequence comprises a topoisomerase recognitionsite and the 3′ sequence comprises an Acc1 restriction site. The oligois exposed to topoisomerase and the topoisomerase binds to 3′ thymidine:

5′ CGAAGGG <CODEA OR B> GTCGACNNNNN 3′  *TTCCC <COMPLEMENT> CAGCTGNNNNN(* = topoisomerase)

DNA is moved to ‘near’ chamber by applying appropriate current anddetecting DNA movement. The topoisomerase-charged ‘codeA’ oligo isprovided in the ‘addition’ chamber. The DNA is moved into the additionchamber by applying appropriate current and detecting DNA movement,whereupon the code A oligo is bound to the DNA, Acc1 is added to the‘reserve’ chamber, where it cleaves at the restriction site to provide atopoisomerase ligation site.

The process is repeated until the desired sequence is reached, addingother ‘code A’ or ‘code B.’ Note that it is not required to continuallyadd new Acc1 to the ‘reserve’ chamber; it is just needed to flush outcodeA or codeB oligos in ‘addition’ chamber when switching from codeA orcodeB.

For sequencing a pore that only allows ssDNA to pass, some modificationsto the protocol above are required. It is already known that when dsDNAencounters a small pore (2 nm) only ssDNA will go through and thecomplement will be ‘stripped’ off. Thus, if doing this synthesis with a2 nm pore one must ensure that the proper dsDNA is able to ‘reform’ onthe other side. To do this one would add “CGAAGGG <CODEA OR B>GTCGACNNNNN” (SEQ ID NO 1) to the near chamber (to ensure a restrictionsite is created) and “CGAAGGG <CODEA OR B> GT” (SEQ ID NO 3) to the farchamber (to ensure a topo-compatible site is generated).

Elaborating on the foregoing method, we demonstrate the sequential‘addition’ of DNA-encoded information into a growing DNA chain with ≥2sequential additions (representing 2 bits of data), each of whichcomprise an ‘add’ and a ‘deprotect’ step. Initial experiments foroptimization and proof of concept are performed in microtubes.

In the approach described in this example, one bit of information isencoded in a string of nucleotides. The DNA bit to be ‘added’ is a shortdsDNA sequence conjugated to vaccinia topoisomerase I (topo). In thepresence of a suitable ‘deprotected’ ‘acceptor’ DNA, the topo-chargedDNA ‘bit’ is enzymatically and covalently linked (‘added’) to theacceptor by the topoisomerase, which in the process becomes removed fromthe DNA. A restriction enzyme can then cleave the added bit to‘deprotect’ it and create of suitable ‘acceptor’ sequence for additionof the next bit.

Topo Charging:

A generic charging scheme is as follows, depicted schematically in FIG.22 and below, where N indicates any nucleotide, and A, T, G, and Crepresent nucleotides with adenine, thymine, guanine and cytosine basesrespectively. N's on top of one another are complementary. While thisexample uses the restriction enzyme HpyCH4III, the basic strategy willwork with other restriction enzymes, e.g., as demonstrated in Example 4.

N-N-N-N-N-N-N-N-N   A-G-G-G-N-N-N-N-N-N-N-N-N-NN-N-N-N-N-N-N-N-N-T-T-C-C-C-N-N-N-N-N-N-N-N-N-N + Topoisomerase * =N-N-N-N-N-N-N-N-N N-N-N-N-N-N-N-N-N +     A-G-G-G-N-N-N-N-N-N-N-N-N-N*-T-T-C-C-C-N-N-N-N-N-N-N-N-N-N (topo charged) (N's on top of oneanother are complementary) Addition Generic ‘add’ reaction:N-N-N-N-N-N-N-N-N-N-A N-N-N-N-N-N-N-N-N-N.. (acceptor) +    A-G-G-G-N-N-N-N-N-N-N-N-N-N *-T-T-C-C-C-N-N-N-N-N-N-N-N-N-N (topocharged) = N-N-N-N-N-N-N-N-N-N-A A-G-G-G-N-N-N-N-N-N-N-N-N-NN-N-N-N-N-N-N-N-N-N-T-T-C-C-C-N-N-N-N-N-N-N-N-N-N + * (free topo)Deprotection Generic ‘deprotection’ reaction:...N-N-A-C-A-G-T-N-N-N-N-N-N-N-N-N-N...N-N-T-G-T-C-A-N-N-N-N-N-N-N-N-N-N + HpyCH4III (restriction enzyme) =...N-N-A-C-A ...N-N-T-G.. (deprotected) + ..G-T-N-N-N-N-N-N-N-N-N-NT-C-A-N-N-N-N-N-N-N-N-N-N (side product)

The following oligonucleotides are ordered from Integrated DNATechnologies (IDT). The “b” at the end of some of the oligonucleotidesindicates biotin):

(SED ID NO 4) BAB: CGATAGTCTAGGCACTGTTTGCTGCGCCCTTGTCCGTGTCGCCCTTATCTACTTAAGAGATCATACAGCATTGCGAGTACG B1:b-CACGTACTCGCAATGCTGTATGATCTCTTAAGTAGATA (SEQ ID NO 5): B2:ATCTACTTAAGAGATCATACAGCATTGCGAGTACG TA1:b-CACACTCATGCCGCTGTAGTCACTATCGGAAT (SEQ ID NO 6): TA2:AGGGCGACACGGACAGTTTGAATCATACCG (SEQ ID NO 7): TA3b:AACTTAGTATGACGGTATGATTCAAACTGTCCGTGTCGCCCTTA TTCCGATAGTGACTACAGCGGCATGAGTB1: b-CACACTCATGCCGCTGTAGTCACTATCGGAAT (SEQ ID NO 8): TB2:AGGGCGCAGCAAACAGTGCCTAGACTATCG (SEQ ID NO 9): TB3b:AACTTAGTATGACGATAGTCTAGGCACTGTTTGCTGCGCCCTTA TTCCGATAGTGACTACAGCGGCATGAG(SEQ ID NO 10): FP1: CACGTACTCGCAATGCT (SEQ ID NO 11): FP2:CGGTATGATTCAAACTGTCCG (SEQ ID NO 12): FP3: GCCCTTGTCCGTGTC

Oligonucleotides are solubilized to 100 uM in TE buffer and stored at−20 C.

Hybridized oligonucleotides are made by mixing oligonucleotides asdescribed below, heating to 95° C. for 5 minutes, and then dropping thetemperature by 5° C. every 3 minutes until the temperature reaches 20°C. Hybridized oligonucleotides are stored at 4° C. or −20° C. Thecombinations of oligonucleotide are as follows:

B1/2

48 uL B1

48 uL B2

4 uL 5M NaCl

A5

20 uL TA1

20 uL TA2

5 uL TA3b

4 uL 5M NaCl

51 uL TE

B5

20 uL TB1

20 uL TB2

5 uL TB3b

4 uL 5M NaCl

51 uL TE

The following buffers and enzymes are used in this example:

-   -   TE: 10M Tris pH 8.0, 1 mM EDTA, pH 8.0    -   WB: 1M NaCl, 10 mM Tris pH8.0, 1 mM EDTA pH8.0    -   1× Topo: 20 mM Tris pH7.5, 100 mM NaCl, 2 mM DTT, 5 mM MgCl₂    -   1× RE: 50 mM K-acetate, 20 mM Tris-acetate, 10 mM Mg-acetate,        100 ug/ml BSA pH 7.9 @ 25 C.    -   Vaccinia DNA Topoisomerase I (topo) is purchased from Monserate        Biotech (10,000 U/mL)    -   HypCH4III is purchased from NEB

Streptavidin-coated magnetic beads (s-MagBeads) are purchased fromThermoFisher.

Acceptor is prepared as follows: 5 uL of s-magbeads are washed one timein 200 uL WB (binding time 1 minute). 5 uL B1/2+195 uL WB is added tobeads and incubated 15 minutes at room temperature, then washed one timewith 200 uL WB, then washed one time with 200 uL 1× Topo, andresuspended in 150 uL of 1× Topo

Topo-charged A5 (see FIG. 20) is prepared as follows: 4 uL 10× topobuffer+23 uL water+8 uL A5+5 uL topo are incubated at 37° C. for 30minutes, added to 5 uL s-magbeads (washed 1× with 200 uL WB, 1× with 200uL 1× Topo, resuspended in 150 uL 1× topo), and allowed to bind for 15minutes at room temperature.

‘Add’ charged A5 to Acceptor: s-magbeads are removed fromTopo-charged-A5, added to Acceptor, and incubated at 37° C. for 60minutes. The aliquot is removed, diluted 1/200 in TE, and stored at −20C

Deprotection: The material is washed one time with 200 uL of WB, whenwashed one time with 200 uL of 1× RE, and resuspended in 15 uL 10× REand 120 uL water). 15 uL HypCH4III is added. The mixture is incubated at37° C. for 60 minutes, then washed one time with 200 uL WB, washed onetime with 200 uL 1× topo, to produce a product which we term‘Acceptor-A5’.

Topo charged B5 (see FIG. 21) is prepared as follows: 4 uL 10× topobuffer. 23 uL water and 8 uL B5+5 uL topo are combined and incubated at37° C. for 30 min. the product is added to 5 uL s-magbeads (washed onetime with 200 uL WB, one time with 200 uL 1× Topo, and resuspended in150 uL 1× topo) and allowed to bind for 15 minutes at room temperature.

‘Add’ charged B5 to Acceptor-A5: s-magbeads are removed fromTopo-charged-B5, added to Acceptor-A5 and incubated at 37° C. for 60minutes. The aliquot is then removed, diluted 1/200 in TE, and stored at−20° C.

Deprotection: The material is washed one time with 200 uL of WB, thenwashed one time with 200 uL of 1× RE, and resuspended in 15 uL 10× REand 120 uL water. 15 uL HypCH4III is added, and the mixture is incubatedat 37° C. for 60 minutes.

Confirmation that the above reactions worked is provided by PCRamplification of aliquots from A5 (Acceptor with A5 added: step iii, ‘A5Added’ in schematic) and B5 (Acceptor-A5 with B5 added: step vi, ‘B5Added’ in schematic). ‘No template’ is used as negative control for A5,A5 is used as negative control for B5, oligo BAB is used as positivecontrol for B5. The expected product size for A5 PCR is 68 bp, theexpected product size for B5 PCR is 57 bp. (B1/2 is also run on the gel,expected size is ˜47 bp, but this may be approximate as there areoverhangs and it is biotinylated). PCR reactions (30 cycles of 95/55/68(1 minutes each) are carried out as follows:

A5 (−) B5 (−) ctrl A5 ctrl B5 Template 1 uL 1 uL 1 uL FP1 1 uL 1 uL 1 uL1 uL FP2 1 uL 1 uL FP3 1 uL 1 uL Water 8 uL 7 uL 7 uL 7 uL Maxima MM 10uL  10 uL  10 uL  10 uL 

SDS-PAGE using 4-20% Tris-glycine gels is used to confirm that expectedsize oligonucleotides are produced. Charging is performed as describedabove, but directly after charging (37° C. incubation step), loadingbuffer is mixed in and samples are heated to 70° C. for 2 minutes andallowed to cool prior to running the gel. Gel is stained with Coomassie.For the negative control, water is added to the reaction instead oftopo. FIG. 30 depicts the results, clearly showing bands correspondingto the expected product sizes for A5 PCR and for B5 PCR.

DNA bit addition via topoisomerase-charged DNA cassettes anddeprotection performed via restriction enzyme are thus shown to befeasible. In these proof of concept experiments the DNA is immobilizedvia streptavidin-conjugated magnetic beads, and moved sequentially intodifferent reaction mixes, but in the nanopore chip format, we createseparate reaction chambers and use electrical current to move the DNAinto those different reaction chambers.

Finally, PCR demonstrates that the expected DNA sequences are createdwhen performing sequential additions of DNA sequences corresponding to‘bits’ of information. These reactions have worked as designed, evenwith minimal optimization.

DNA made as described in examples 2 and 3 is recovered and sequenced,using a commercial nanopore sequencer (MinION from Oxford Nanopore),confirming that the desired sequence is obtained.

Example 4: DNA Synthesis: Block Oligonucleotide Addition, Using aDifferent Restriction Enzyme

The following synthesis is carried analogously to Example 3, but usingthe restriction enzyme MluI, which cuts at ‘ACGCGT’ to form:

...NNNA     CGCGTNNN... ...NNNTGCGC     ANNN...In this example TOPO is charged to form a complex with sequencecomplementarity that will enable the charged TOPO to transfer DNA to DNAcut with MluI:

5′ pCACGTCAGGCGTATCCATCCCTTCGCGTTCACGTACTCGCAATGCTGTAG3′  GTGCAGTCCGCATAGGTAGGGAAGCGC AGTGCATGAGCGTTACGAGATCb + TOPO =5′ pCACGTCAGGCGTATCCATCCCTT*          ( 3′  GTGCAGTCCGCATAGGTAGGGAAGCGC(‘*’ indicates TOPO bound at 3′ phosphate) + CGCGTTCACGTACTCGCAATGCTGTAG     AGTGCATGAGCGTTACGAGATCb (b = biotin. This can be removed withstreptavidin)

By a process analogous to the preceding example, the charged TOPO isthen used to add the oligomer to the 5′ end of strand being synthesized,having a complementary acceptor sequence, thereby releasing the TOPO,and the strand is then “deprotected” using the WA, and the cyclerepeated until the desired sequence of oligomers is obtained.

Example 5: Addition of Single Base Using Topoisomerase Strategy

We have found that the topoisomerase system can also be designed to addsingle bases to a single stranded DNA chain (in comparison to Example 3,which describes adding ‘cassettes’). The DNA bit to be ‘added’ iscontained in a short DNA sequence conjugated to vaccinia topoisomerase I(topo). In the presence of a suitable single stranded ‘deprotected’‘acceptor’ DNA, the topo-charged DNA is enzymatically and covalentlyligated (‘added’) to the acceptor by the topoisomerase, which in theprocess becomes removed from the DNA. A type IIS restriction enzyme canthen cleave all of the added DNA with the exception of a single base(the base which is being ‘added’). This process of deprotect-add isrepeated to add additional bases (bits).

Topo Charging: A generic charging protocol is as follows, similar toExample 3:

...N-N-N-N-N-N-N-N-N-C-C-C-T-T-N-N-N-N-N-N-N-N-N-N-N-N-N......N-N-N-N-N-N-N-N-N-N-N-N-N-N-I-I-I-I-I   N-N-N-N-N-N-N...biotin+ Topoisomerase (*) = N-N-N-N-N-N-N-N-N-N-N-N-N-N-NN-N-N-N-N-N-N-N-N...biotin (by-product) +...N-N-N-N-N-N-N-N-N-C-C-C-T-T* ...N-N-N-N-N-N-N-N-N-N-N-N-N-N-I-I-I-I-I(topo charged)As in Example 3, the N's on top of one another are complementary. I isinosine. The biotin is used to remove unreacted product and byproduct.Addition of a single base is carried out as follows

N-N-N-N-N-N-N-N-N-N... (acceptor sequence nucleotides indicated initalics) + ...N-N-N-N-N-N-N-N-N-C-C-C-T-T*...N-N-N-N-N-N-N-N-N-N-N-N-N-N-I-I-I-I-I (topo charged) =...N-N-N-N-N-N-N-N-N-C-C-C-T-T-N-N-N-N-N-N-N-N-N-N......N-N-N-N-N-N-N-N-N-N-N-N-N-N-I-I-I-I-I + * (free topo)Deprotection is illustrated as follows, using BciVI restriction enzyme(site in bold):

...N-G-T-A-T-C-C-N-N-C-C-C-T-T-N-N-N-N-N-N-N-N-N-N......N-N-N-N-N-N-N-N-N-N-N-N-N-N-I-I-I-I-I + BciVI (restriction enzyme) =T-N-N-N-N-N-N-N-N-N-N... (note a ‘T’ has been added to the 5′ of theacceptor DNA) + N-N-I-I-I-I-I (dissociated*) +...N-G-T-A-T-C-C-N-N-C-C-C-T ...N-N-N-N-N-N-N-N-N-N-N-N------------------------------------------------------------T-N-N-N-N-N-N-N-N-N-N...   N-N-I-I-I-I-I = T-N-N-N-N-N-N-N-N-N-N...+ N-N-I-I-I-I-I (NNIIIII dissociates from the single strand with addedbase)

The following oligonucleotides are synthesized commercially (B=biotin,P-phosphate, I=inosine):

(SEQ ID NO 13): NAT1: CACGTCAGGCGTATCCATCCCTTCACGTACTCGCAATGCTGTATGGCGAT NAT1b P-CACGTCAGGCGTATCCATCCCTTCACGTACTCGCAATGCTGT ATGGCGAT-B(SEQ ID NO 31): NAT9cI: P-IIIIIAAGGGATGGATACGCCTGACGTG (SEQ ID NO 14):NAT9x: P-ATCGCCATACAGCATTGCGAG (SEQ ID NO 15): NAT9:ACGTGAAGGGATGGATACGCCTGACGTG (SEQ ID NO 16): Nat9Acc:CACGTAGCAGCAAACAGTGCCTAGACTATCG (SEQ ID NO 17): Nat1P:CACGTCAGGCGTATCCATCC (SEQ ID NO 18): FP4: CGATAGTCTAGGCACTGTTTG

The oligonucleotides are solubilized to 100 μM in TE buffer and storedat −20° C.

Hybridization: The following hybridized oligonucleotides are made bymixing the oligonucleotides as described, heating to 95° C. for 5minutes, and then dropping the temperature by 5° C. every 3′ until thetemperature reaches 20° C. Hybridized oligonucleotides are stored at 4°C. or −20° C.

NAT1b/NAT9cI/NAT9x

8 μL NAT1B

10 μL NAT9cI

10 μL NAT9x

48 μL TE

4 μL 5M NaCl

NAT1/NAT9cI

10 μL NAT1

10 μL NAT9cI

80 uL PBS

NAT1/NAT9

10 μL NAT1

10 μL NAT9

80 uL PBS

Buffers & Enzymes: The following buffers are used:

-   -   TE: 10M Tris pH 8.0, 1 mM EDTA, pH 8.0    -   PBS: phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM        Na₂HPO₄, 1.8 mM KH₂PO₄)(pH 7.4)    -   10× Cutsmart: 500 mM KAc, 200 mM Tris-Ac, 100 mM Mg-Ac, 1 mg/mL        BSA pH 7.9        BciVI is purchased from NEB and Streptavidin-coated magnetic        beads (s-MagBeads) are purchased from ThermoFisher

The addition reaction is carried out as follows.

1. Topo charge: The reagents are assembled as per table:

(−) control (−) control Experiment #1 #2 10x topo buffer 3 3 3 Water 1721  23  NAT1B/NAT9cI/NAT9x 6 6 — Topo 4 — 4The reagents are then incubated at 37° C. for 30 minutes. The byproductsare removed using streptavidin magnetic beads (5 uL) in 1× topo bufferafter 10 minutes at room temperature to allow binding.

2. Reaction: The reagents are assembled as per table:

(−) control (−) control Experiment #1 #2 From A.1.c 1 1 1 NAT9Acc 1 1 110x topo buffer 1 1 1 water 7 7 7The reagents are then incubated at 37° C. for 30 minutes. The additionreaction is expected to proceed as follows:

NAT1B  5′ p-CACGTCAGGCGTATCCATCCCTTCACGTACTCGCAATGCTGTATGGCGAT-B       +TOPO = NAT9cI 3′  GTGCAGTCCGCATAGGTAGGGAAIIIII GAGCGTTACGACATACCGCTA-p NAT9x        5′CACGTCAGGCGTATCCATCCCTT*     CACGTATTCGCAATGCTGTATGGTGAT-B        3′GTGCAGTCCGCATAGGTAGGGAAIIIII        GAGUGTTACGACATACUGCTA

The asterisk (*) represents topoisomerase. Note that NAT9cI isphosphorylated, but this isn't shown for illustration purposes.

When the charged topo is in the presence of an acceptor sequence, itundergoes the following reaction:

5′ p-CACGTCAGGCGTATCCATCCCTT*      GTGCAGTCCGCATAGGTAGGGAAIIIII      +                            CACGTAGCAGCAAACAGTGCCTAGACTATCG      = 5′p-CACGTCAGGCGTATCCATCCCTTCACGTAGCAGCAAACAGTGCCTAGACTATCG     GTGCAGTCCGCATAGGTAGGGAAIIIII

PCR amplification and measurement of the molecular weights of theproduct on agarose gel confirms the expected product is produced. SeeFIG. 30, depicting correct sized band in lane 1 (experiment), no bandsin negative controls.

B. Deprotection Reaction: The reagents are assembled as per table:

1 2 3 4 NAT1/NAT9 1 1 — — NAT1/NAT9cI — — 1 1 10x cutsmart 2 2 2 2 water17  16  17  16  BciVI 0 1 0 1The reagents are incubated at 37° C. for 90 minutes. For thedeprotection reaction, a representative product of an addition reactionis created using purchased oligonucleotides, and tested for digestionwith the BciVI restriction enzyme:

(SEQ ID NO 13): NAT1 5′CACGTCAGGCGTATCCATCCCTTCACGTACTCGCAATGCTGTATGGCGAT + BciVI = NAT9cI 3′GTGCAGTCCGCATAGGTAGGGAAIIIII (SEQ ID NO 13): NAT1 5′CACGTCAGGCGTATCCATCCCT     TCACGTACTCGCAATGCTGTATGGCGAT NAT9cI 3′GTGCAGTCCGCATAGGTAGGG   AAIIIIIIt was not known whether the restriction enzyme would cut the DNA asintended, given that 3′ of the cut site are a series of inosines asopposed to ‘regular’ bases. As a positive control, the ‘appropriately’base-paired equivalent of NAT1/NAT9cI is made (NAT1/NAT9c):

(SEQ ID NO 13): NAT1 5′ CACGTCAGGCGTATCCATCCCTTCACGTACTCGCAATGCTGTATGGCGAT (SEQ ID NO 19): NAT9c 3′ GTGCAGTCCGCATAGGTAGGGAAGTGCAPCR amplification of the product followed by measurement of molecularweight on agarose gels (FIG. 31) shows that the enzyme works asintended. For the positive control, a larger band is observed whenundigested (lane 1), but a smaller band/s are observed with digestion.The same pattern is observed with NAT1/NAT9cI, showing that the presenceof inosines does not negate or interfere with digestion. A small amountof undigested product seems to remain with NAT1/NAT9cI, suggesting thatthe cleavage is not as effective, at least under these conditions, aswith NAT1/NAT9c. Cleavage efficiency may be improved by altering bufferconditions and/or addition of more inosines at the 5′ end of NAT9cI.

The foregoing example demonstrates that it is feasible to use aTopo/TypeIIS restriction enzyme combination to add a single nucleotideto the 5′ end of a target single stranded DNA. A related topoisomerase,SVF, that recognizes the sequence CCCTG(https://www.nebi.nlm.nih.gov/pubmed/8661446) is used to add a ‘G’instead of a ‘T’, using an analogous process, thus allowing constructionof a sequence encoding binary information with T and G.

As noted above, where dsDNA is generated using topoisomerase strategies,nicks in DNA on the opposing strand can be repaired using a ligasetogether with ATP. But when doing the single nucleotide addition, as inthis example, we are building a single stranded DNA, so there are nonicks that need to be repaired and no need to use ligase.

Example 6: Addition of Single Base Using Topoisomerase Strategy Couplewith 5′ Phosphate Coupling

In another approach to single base addition, we use a 5′phosphate as ablocking group to provide single base pair addition in the 3′ to 5′direction. The charging reaction charges the topoisomerase with a singleT (or G, or other nucleotide as desired), having a 5′ phosphate group.When the charged topoisomerase ‘sees’ a free 5′ unblocked(unphosphorylated) single stranded DNA chain it will add the T to thatchain, providing a DNA with a T added to the 5′. This addition isfacilitated by the presence of an adapter DNA having sequences to whichthe topoisomerase and the single stranded acceptor DNA can bind. (Notethat the adapter DNA is catalytic—it can be reused as a template inrepeated reactions.) The added nucleotide has a 5′ phosphate on it, soit won't be a substrate for further addition until it is exposed to aphosphatase, which removes the 5′ phosphate. The process is repeated,using Topo to add a single “T” to the 5′ end of a target single strandedDNA and SVF topoisomerase to add a single ‘G’, thus allowingconstruction of a sequence encoding binary information with T and G. Theprocess is depicted schematically as follows:

GENERICALLY: CHARGING:N-N-N-N-N-N-N-N-C-C-C-T T-N-N-N-N-N-N-N-N (T is 5′ phosphorylated) +TOPO = N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-NN-N-N-N-N-N-N-N-C-C-C-T   N-N-N-N-N-N-N-N + T-TOPO (T is 5′phosphorylated) N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N TRANSFERT-TOPO (T is 5′ phosphorylated) + N-N-N-N-N-N-N-N-N-N-N-N (5′ N has 5′OH) = T-N-N-N-N-N-N-N-N-N-N-N-N (T is 5′ phosphorylated) + TOPODEBLOCKING: T-N-N-N-N-N-N-N-N-N-N-N-N (T is 5′ phosphorylated) +Phosphatase = T-N-N-N-N-N-N-N-N-N-N-N-N (T is 5′dephoaphorylated, now has 5′ OH)*****ALTERNATE TRANSFER MECHANISM************* T-TOPO (T is 5′phosphorylated) + N-N-N-N-N-N-N-N-N-N-N-N (acceptor) (5′ N has 5′ OH) +N-N-N-N-N-N-N-C-C-C-T = TOPO + N-N-N-N-N-N-N-N-N-N-N-A-I-I-I-I-I(adapter) N-N-N-N-N-N-N-C-C-C-T T-N-N-N-N-N-N-N-N-N-N-N-NN-N-N-N-N-N-N-N-N-N-N-A-I-I-I-I-Ithis transient intermediate that breaks down to -->T-N-N-N-N-N-N-N-N-N-N-N-N (T is 5′ phosphorylated) +N-N-N-N-N-N-N-C-C-C-T N-N-N-N-N-N-N-N-N-N-N-A-I-I-I-I-I

Example 7: Using DNA Origami to Aid in Attaching DNA Adjacent toNanopore

A DNA strand with a large origami structure on one end is captured in ananopore, and immobilized to surface-conjugated streptavidin through aterminal biotin moiety on the DNA. After restriction enzyme cleavage ofthe origami structure, the immobilized DNA can be moved back and forththrough the pore, as confirmed by current disruption. The immobilizationenables a controlled movement of a single DNA molecule through the pore,which in turn enables both the ‘reading’ and ‘writing’ of information toDNA.

As depicted in FIG. 35, a bulky double-stranded DNA unit is formed,which is too large to fit through the nanopore, with a single strandedregion, linked to the bulky portion by two short double stranded regionshaving which serves to anchor the DNA to be added to in the synthesis.The single stranded region can then be detached and anchored to thesurface adjacent to the nanopore, and the origami structure released.See FIG. 33.

Nanopores are formed in 3 mm chips with 20 nm SiO2, with 50*50 μmwindows. Chip are provided by Nanopore Solutions. Nanopore cassetteholders and flow cells are provided by Nanopore Solutions. The amplifieris a Tecella Pico 2 amplifier. This is a usb-powered amplifier that usesa usb-computer interface for control. Tecella supplies (Windows)software to control the amplifier. The multimeter is a FLUKE 17B+Digital Multimeter, capable of detecting current as low as 0.1 uA. Forscreening of radiofrequency noise we use a Concentric TechnologySolutions TC-5916A Shield Box (Faraday Cage) with USB interface.Oligonucleotides are obtained from IDT.com. “PS” is ProparylSilane—O-(PROPARGYL)-N-(TRIETHOXYSILYLPROPYL) CARBAMATE fromhttp://www.gelest.com/product/o-propargyl-n-triethoxysilylprorylcarbamate-90/.

The origami structure is based on single-stranded m13 with a ‘honeycomb’cube origami structure which is ˜20 nm on one side. There are doublestranded regions adjacent to the honeycomb each containing a uniquerestriction site. One of those sites is used to attach modified DNA toenable attachment near the nanopore, the other is used for cleaving offthe origami structure once the DNA is attached.

Nanopore Formation:

Nanopores are formed in the chips using dielectric breakdown, asfollows:

-   -   1. Chips are carefully mounted in the cassettes    -   2. Wetting: 100% ethanol is carefully pipetted on the chip.        Bubbles must be removed. However, direct pipetting of solution        on the chip should be avoided or the chip can crack (SiO2 is        only 20 nm).    -   3. Surface treatment: ethanol is removed, and freshly prepared        Piranah solution (75% sulfuric acid, 25% hydrogen peroxide        (30%)) is pipetted onto the chip. (let piranha solution come to        room temperature). Leave on for 30 minutes.    -   4. Rinse 4 times with distilled water.    -   5. Rinse 2 times with HK buffer (10 mM HEPES pH 8, 1M KCl)    -   6. Assemble cassette into flow cell.    -   7. Add 700 μL HK buffer to each chamber of the flow cell.    -   8. Insert silver electrodes attached to the amplifier and close        the Faraday cage.    -   9. Test resistance with 300 mV. No current should be detected.        If it is detected, the chip is likely cracked and one must start        again.    -   10. Connect electrodes to a DC current of 6 V and test the        current with a multimeter. Current should be low and should not        change. Increase voltage by 1.5 V and hold the voltage until the        resistance increases. If resistance does not increase after 5-10        minutes, increase the voltage another 1.5 V and try again.        Repeat until resistance increases, at which point the applied        voltage should be stopped immediately. (with sufficient voltage,        dielectric breakdown occurs and a ‘hole’ is created in the SiO2        membrane. When initially created the hole is small, but will        increase in size as the voltage is maintained.)    -   11. Test the pore using the amplifier. At 300 mV one should see        current of a few to several nA. The more current, the larger the        pore.

FIG. 34 depicts a basic functioning nanopore. In each panel, the y-axisis current (nA) and the x-axis is time (s). The left panel “Screening ofRF Noise” illustrates the utility of the Faraday cage. A chip with nonanopore is placed in the flow cell and 300 mV applied. When the lid ofthe Faraday cage is closed (first arrow) the noise reduction can beseen. A small spike occurs when the latch is closed (second arrow).Notice the current is ˜0 nA. After pore manufacture (middle panel),application of 300 mV (arrow) results in a current of ˜3.5 nA. When DNAis applied to the ground chamber and +300 mV is applied DNAtranslocations (right panel) can be observed as transient decreases inthe current. (Note, in this case the TS buffer is used: 50 mM Tris, pH8, 1M NaCl). Lambda DNA is used for this DNA translocation experiment.

Silver Chloride Electrodes:

-   -   1. Silver wire is soldered to insulated copper wire.    -   2. Copper wire is grounded, and silver is dipped into fresh 30%        sodium hypochlorite for 30 minutes.    -   3. Silver should acquire a dark gray coating (silver chloride).    -   4. Silver wire is rinsed extensively in distilled water and        dried.    -   5. It is now ready for use.

Silanization of Beads:

The silanization method is initially developed/tested on SiO₂ coatedmagnetic beads (GBioscience). The following protocol is adopted:

1. Pretreat beads in fresh pirannah solution for 30 minutes.

2. Wash 3× with distilled water.

3. Wash 2× in methanol.

4. Dilute APTES stock 1:500 in methanol.

5. Add diluted APTES to beads, incubate at RT for 45 minutes.

6. Rinse with methanol.

7. 100° C. for 30 minutes.

8. Store under vacuum.

Silanization of Silicon Chip

-   -   1. Mount chip with nanopore in a cassette.    -   2. Rinse with methanol, carefully removing any air bubbles.    -   3. Add fresh pirannah solution (equilibrated to room        temperature) and incubate for 30 minutes.    -   4. Wash 4× with distilled water.    -   5. Wash 3× with methanol.    -   6. Dilute APTES stock 1:500 in methanol and use to wash chip 2×.        Incubate at RT for 45 minutes.    -   7. Rinse 2× with methanol.    -   8. Dry under and air stream.    -   9. Store under vacuum overnight.

Streptavidin Conjugation of Beads:

The streptavidin conjugation is initially developed/tested on thesilanized beads prepared above.

-   -   1. Wash silanized beads with Modified Phosphate-Buffered Saline        (MPBS)    -   2. Make a fresh solution of 1.25% glutaraldehyde in MPBS (using        50% glutaraldehyde stock, stored frozen).    -   3. Add 1.25% glutaraldehyde to beads and let stand for 60′ with        gentle up-down pipetting every 15 minutes.    -   4. Wash 2× with MPBS.    -   5. Wash 2× with water.    -   6. Let dry under vacuum.    -   7. Add streptavidin (500 m/mL in MPBS) to beads and incubate 60        minutes. (For negative control beads, use bovine serum albumin        (BSA) (2 mg/mL in MPBS) in place of streptavidin).    -   8. Remove streptavidin and add BSA (2 mg/mL in MPBS). Incubate        60 minutes.    -   9. Wash 2× in MPBS.    -   10. Store at 4° C.

Streptavidin Conjugation of Silicon Chip

-   -   1. Rinse silanized chip with ethanol 2×    -   2. Rinse silanized chip with MPBS 2×    -   3. Make a fresh solution of 1.25% glutaraldehyde in MPBS (using        50% glutaraldehyde stock, stored frozen).    -   4. Rinse chip with 1.25% glutaraldehyde 2×, let stand for 60′        with gentle up-down pipetting every 15 minutes    -   5. Wash 2× with MPBS    -   6. Wash 2× with water    -   7. Let dry under air stream    -   8. To one half of the chip add BSA (2 mg/mL in MPBS), and to the        other add streptavidin (500 μg/mL in MPBS). Incubate 60 minutes.        Make a marking on the cassette to indicate which half of the        chip is streptavidin modified.    -   9. Rinse both halfs of the chip with BSA (2 mg/mL in MPBS).        Incubate 60 minutes.    -   10. Wash in MPBS.

The buffers used herein are made as follows:

-   -   MPBS: 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L disodium-phosphate,        0.240 g/L potassium phosphate, 0.2 g/L polysorbate-20 (pH 7.2)    -   PBS: 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L disodium-phosphate, 0.240        g/L potassium phosphate    -   TS: 50 mM Tris pH 8.0, 1M NaCl    -   HK: 10 mM HEPES pH 8.0, 1M KCl    -   TE: 10 mM Tris, 1 mM EDTA, pH 8.0    -   Pirannah Solution: 75% hydrogen peroxide (30%)+25% sulfuric acid    -   APTES stock: 50% methanol, 47.5% APTES, 2.5% nanopure water. Age        at 4° C. for at least 1 hour. Store at 4° C.    -   PDC stock: 0.5% w/v 1,4-phenylene diisothiocyanate in DMSO

Oligonucleotides (5′ TO 3′) are ordered:

(SEQ ID NO 20): o1 CTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATG(SEQ ID NO 21): o3 GGAAAGCGCAGTCTCTGAATTTAC (SEQ ID NO 22):N1 CTTACTGGAACGGCTATCGATATCGCAGCAGGACAGABN1 Biotin-CTTACTGGAACGGCTATCGATATCGCAGCAGGACAGA (SEQ ID NO 23):N2 GTCCTGCTGCGATATCGATAGCCGTTCCAGTAAG

Oligonucleotide pair hybridization is carried out as follows:

1. Make stock solutions of oligo's at 100 uM concentration in TE buffer

2. Dilute oligos to 10 μM in PBS

3. Heat to 85° C. for 5′ in a thermal cycler

4. Ramp heat down by 5° C. every 3′ until 25° C.

5. Store at 4° C. or −20° C.

Streptavidin Conjugation:

Streptavidin conjugation to SiO₂ is developed and tested using SiO2coated magnetic beads, and the protocols were then adapted for SiO₂chips. Binding of biotinylated oligos to both streptavidin and BSAconjugated beads are tested. As expected, negligible binding is observedwith BSA-conjugated beads, while strong binding is observed withstreptavidin conjugated beads. See FIG. 38. Since it would be moreconvenient to perform the conjugation in high salt (DNA movement isperformed in high salt), the ability of the beads to bind in HK bufferis also tested. Binding in HK buffer is comparable to binding in MPBSbuffer (FIG. 39).

Origami constructs are made and confirmed operable as described above inFIGS. 35-37. Biotinylation of the origami structure is tested usingoligonucleotides. We already know from the ‘Orgami’ results describedabove for FIG. 37 that the AlwNI site is active. An oligonucleotide pairthat recreates a segment of the exact sequence in the origami DNA isused below (o1/o3). The origami molecule is depicted in FIG. 77:

The oligo pair o1/o3 is

(SEQ ID NO 20) CTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATG o1         CATTTAAGTCTCTGACGCGAAAGG o3 (SEQ ID NO 21)

The DNA is digested with AlwNI in the presence of T4 DNA ligase, and abiotinylated oligo that is complementary to the overhang on the 3′ sideof the origami sequence (which itself is attached to a long ssDNAsequence which itself is attached to the other side of the origami),according to the following reaction:

CTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATG o1 (SEQ ID NO 20) +A1wNI =        CATTTAAGTCTCTGACGCGAAAGG                o3 (SEQ ID NO 21)CTGGAACGGTAAATTCAGAGA     CTGCGCTTTCCATTCTGGCTTTAATG (SEQ ID NO 24) +        CATTTAAGTC       TCTGACGCGAAAGG (SEQ ID NO 25)B-CTTACTGGAACGGCTATCGATATCGCAGCAGGACAGA         BN1 (SEQ ID NO 22) +ligase =  GAATGACCTTGCCGATAGCTATAGCGTCGTCCTG            N2  (SEQ ID NO 23)B-CTTACTGGAACGGCTATCGATATCGCAGCAGGACAGACTGCGCTTTCCATTCTGGCTTTAATG  GAATGACCTTGCCGATAGCTATAGCGTCGTCCTGTCTGACGCGAAAGGIn this strategy, AlwN1 cleaves the target DNA. When the ligase is addedit is possible for this DNA to be religated, but the restriction enzymewill cut it again. However, if/when the (right) fragment (of o1/o3)binds to BN1/N2, the restriction site is NOT recreated, thus thisproduct will not be cut. Specific attachment is confirmed, by testingwith and without the restriction enzyme:

1 2 o1/o3 1 1 n1/bn2 1 1 10x lig buf 2 2 water 15 14.5 AlwNI .5 Ligase 11All reagents except ligase are added and solution is incubated at 37° C.for 60 minutes. Ligase is added and solution incubated overnight at 16°C. 10× lig buff refers to NEB 10× T4 DNA ligase buffer. Ligase in NEB T4DNA ligase. o1/o3 and n1/n2 refer to annealed oligo pairs, as depictedabove. Units are microliters. Agarose gel analysis confirms that in thepresence of the AlwNI, a larger product is formed, corresponding to thebiotinylated oligonucleotide attached to the long ssDNA arm attached tothe origami structure. A similar strategy is used for 3′ biotinylation,where desired.

Above we demonstrate the ability to form and use a nanopore to detectvoltage induced transit of DNA across the pore, the creation of anorigami molecule with a long ss region attached at its' far end to abiotin, and the conjugation of streptavidin to silicon dioxide, and touse that to capture biotinylated DNA. These tools are used to attach andcontrol the movement of a single DNA molecule next to a nanopore.

The first step is to conjugate streptavidin to one surface of an SiO2nanopore (and BSA to the other side). This is accomplished according tothe protocol above. The resulting pores tend to have a lower currentthan they initially have. After some brief 6v pulses, the currentsreturn to be near their original current. A functioning nanopore at thispoint is shown in FIG. 40.

Next, the origami DNA is inserted. When the origami DNA is added to theappropriate chamber and the current turned on, the origami will insertinto the chamber. A representation of this is shown in FIG. 41.Experimental results when the origami is introduced at a finalconcentration of 50 pM confirm that the DNA with the origami insertsinto the pore relatively soon (typically in seconds), which isdetectable by the resulting reduction of current flow across thenanopore (e.g., in these experiments, current before origami insertionis ˜3 nA, and ˜2.5 nA after insertion). If the current is allowed to runfor longer times, double insertions can be observed. If higherconcentrations are used, insertion occurs too quickly to be observed.

Binding of Inserted DNA to Chip.

After the origami is inserted into the nanopore, 15 minutes are allowedto elapse before voltage is applied again. The end of the ssDNA regionof the origami contains a biotin, and streptavidin is conjugated to thesurface of the nanopore. Streptavidin binds to avidin with an affinityconstant that approaches that of a covalent bond. The 15 minute timeallows the DNA to diffuse and for the biotin end to find and bind to thestreptavidin. If the DNA has in fact become attached to the surface,when the voltage is reversed the observed current should be slightlyless than what was seen previously. Also, switching the current back andforth should result in currents in both directions that are lower thanthat seen with a free pore. In the example shown here, a free pore showsa current of ˜3 nA. FIG. 42 shows a representation of attached DNA, andFIG. 43 shows experimental results of voltage switching the attachedorigami DNA. Note that the currents seen in both directions are ˜+/−2.5nA, which is lower than the ˜+/−3 nA observed with a free pore. If theDNA hasn't bound to the surface, the original current will be recoveredwhen the voltage is switched (FIG. 44).

In order to remove the origami structure, the buffer in the flow cellchamber containing the origami structure was removed and replaced with1× Swa1 buffer with 1 uL Swa1/20° L. The buffer in the other flow cellchamber is replaced with 1× Swa1 buffer without Swa1. This is incubatedat room temperature for 60 minutes, then washed with HK buffer, andvoltage applied. Movement of the DNA back and forth as represented inFIG. 45 is confirmed by the experimental data in FIG. 46, showingcontrolled movement of immobilized DNA through a SiO2 nanopore.

Example 8—Alternative Means to Attach the Polymer to the SurfaceAdjacent to the Nanopore

The foregoing examples describe attachment of DNA to the surfaceadjacent to the nanopore by biotinylating the DNA and coating theattachment surface with streptavidin. Some alternative means ofattaching the polymer are depicted in FIG. 47.

a) DNA Hybridization:

In one method, the DNA which is extended in the methods of the inventionis hybridized to a short oligonucleotide which is attached near thenanopore. Once the synthesis is complete, the synthesized DNA can beeasily removed without a need for restriction enzymes, or alternativelythe double strand formed by the bound oligonucleotide and thesynthesized DNA can provide a substrate for a restriction enzyme. Inthis example, the short oligomers are conjugated to the surface usingbiotin-strepavidin, or ligated using 1,4-phenylene diisothiocyanate asfollows:

Conjugation of biotinylated DNA to SiO2:

-   -   A. SILANIZE:    -   1. Pre-treatment: nha solution for 30 minutes, wash with double        distilled H₂O (ddH₂O)    -   2. Prepare APTES stock: 50% MeOH, 47.5% APTES, 2.5% nanopure        H₂O: age>1 hr 4° C.    -   3. dilute APTES stock 1:500 in MeOH    -   4. incubate chips at room temperature    -   5. rinse MeOH    -   6. dry    -   7. heat at 110° C. for 30 minutes

CONJUGATE:

-   -   1. Treat chip with PDC stock 5h (room temperature) (PDC stock:        0.5% w/v 1,4-phenylene diisothiocyanate in DMSO)    -   2. 2 washes in DMSO (brief!)    -   3. 2 washes in ddH₂O (brief!)    -   4. 100 nM amino-modified DNA in ddH₂O (pH 8) O/N 37° C.    -   5. 2 washes 28% ammonia solution (deactivate)    -   6. 2 washes ddH₂O

Single stranded DNA having a terminal sequence complementary to theattached oligonucleotides is introduced as described above and allowedto hybridize with the attached oligonucleotides.

b) Click Chemistry:

Click chemistry is a general term for reactions that are simple andthermodynamically efficient, do not create toxic or highly reactivebyproducts, and operate in water or biocompatible solvents, and areoften used to join substrates of choice with specific biomolecules. Theclick conjugation in this case uses similar chemistry as used in a) toattach the oligonucleotides, only it is here used to attach the polymerwhich is extended in the course of synthesis in the methods of theinvention. While in this example, DNA is the polymer, this chemistrywould work to attach other polymers which have been functionalized byaddition of a compatible azide group.

SILANIZE:

-   -   1. Pre-treatment: piranha solution for 30′, wash ddH2O    -   2. Prepare PS (propargyl silane) stock: 50% MeO H, 47.5% PS,        2.5% nanopure H2O: age>1 hr 4 C    -   3. Dilute APTES stock 1:500 in MeOH    -   4. Incubate chips at room temperature    -   5. Rinse MeOH    -   6. Dry    -   7. Heat at 110° C. for 30 minutes

DNA which is terminated in an azide functional group will covalentlybind to this surface (as shown in FIG. 47). Azide terminated oligos areordered, and attached to the longer origami DNA, as described for thebiotin addition to the DNA previously.

Example 9: Optimized Topoisomerase-Mediated DNA Synthesis

An oligonucleotide cassette is composed of three oligonucleotides thatare hybridized to form a double-stranded DNA cassette. The cassettes aredesigned with a vaccinia virus topoisomerase recognition sequence(CCCTT) on the plus DNA strand and followed by a GCCG sequence on theminus strand. Upon topoisomerase recognition of its target sequence, thetopoisomerase cleaves the oligonucleotide, and forms a covalent bondwith the 5′ section of the oligonucleotide, resulting in the formationof a “charged” topoisomerase. The addition of unmatched base pairs just3′ of the CCCTT on the plus strand (CGAA matched to the GCCG on theminus strand) results in more efficient topoisomerase charging. Thecleaved 3′ portion of the oligonucleotide cassette (termed by-product)can be removed from the charged topoisomerase when streptavidin-coatedbeads are added to the mixture, binding to the biotin attached to the 3′end of the plus DNA strand. This reaction is depicted as follows:

Initial Charging Oligonucleotide:

5′ GCGCACGGTCTCCCGGCGTATCCATCCCTTCGAATTCACGTACTCGCCAGTCTACAG-biotin 3′3′ CGCGTGCCAGAGGGCCGCATAGGTAGGGAAGCCG AGTGCATGAGCGGTCAGATGTC        5′The oligonucleotide in italics is 5′ phosphorylated.After Topoisomerase Charges:

5′ GCGCACGGTCTCCCGGCGTATCCATCCCTT       3′ (SEQ ID NO 26) 3′CGCGTGCCAGAGGGCCGCATAGGTAGGGAAGCCG   5′Topoisomerase is covalently linked the 3′ T of the CCCTT recognitionsequence (not shown).The by-product oligonucleotide is removed by the biotin bound tostreptavidin beads

5′ CGAATTCACGTACTCGCCAGTCTACAG-biotin 3′bound to S/A coated magnetic beads 3′     AGTGCATGAGCGGTCAGATGTC        5′The charged topoisomerase has the unique ability to add the 5′ sectionof the oligonucleotide to a DNA acceptor strand that has a complementaryoverhang:

DNA bound to the Charged Topo (SEQ ID NO 26): DNA Acceptor Strand (SEQ ID NO 27):5′ GCGCACGGTCTCCCGGCGTATCCATCCCTT     3′ +

CTCGCAATGCTGTATGGCGATGGAATTCCACAGTCAGCAG 3′CGCGTGCCAGAGGGCCGCATAGGTAGGGAAGCCG 5′CAGCGTTACGACATACCGCTACCTTAAGGTGTCAGTCGTCThe DNA acceptor strand extended by one cassette (or bit):

(SEQ ID NO 28): 5′ GCGCACGGTCTCCCGGCGTATCCATCCCTT

CTCGCAATGCTGTATGGCGATGGAATTCCACAGTCAGCAG 3′ 3′CGCGTGCCAGAGGGCCGCATAGGTAGGGAAGCCGGAGCGTTACGACATACCGCTACCTTAAGGTGTCAGTCGTC 5′The topoisomerase is released by this reaction. The oligonucleotidecassette, or bit, is then “deprotected” by digestion with therestriction enzyme Bsa I, which recognizes the nucleotides GGTCTC. Bsa Iis a type IIS restriction enzyme, which recognizes asymmetric DNAsequences (the GGTCTC sequence) and cleaves outside of their recognitionsequence. The cassette was designed so that cleavage with Bsa I willresult in a CGGC overhang. This overhang, the same as is found in theDNA acceptor strand, allows another cassette, or bit, to be added byanother charged topoisomerase.

(SEQ ID NO 28): DNA acceptor with one protected cassette (Bsa I restriction site in bold):5′GCGCACGGTCTCCCGGCGTATCCATCCCTTCGGCCTCGCAATGCTGTATGGCGATGGAATTCCACAGTCAGCAG 3′3′CGCGTGCCAGAGGGCCGCATAGGTAGGGAAGCCGGAGCGTTACGACATACCGCTACCTTAAGGTGTCAGTCGTC 5′(SEQ ID NO 29): DNA acceptor with one cassette after Bsa I digestion: 5′CGGCGTATCCATCCCTTCGGCCTCGCAATGCTGTATGGCGATGGAATTCCACAGTCAGCAG 3′ 3′    CATAGGTAGGGAAGCCGGAGCGTTACGACATACCGCTACCTTAAGGTGTCAGTCGTC 5′Incubation with charged topoisomerase results in the addition of anothercassette (bit), and extends the DNA chain to encode more information.

DNA bound to the Charged Topo DNA Acceptor strand plus one cassette(SEQ ID NO 26): (SEQ ID NO 29): 5′ GCGCACGGTCTCCCGGCGTATCCATCCCTT +CGGCGTATCCATCCCTTCGGCCTCGCAATGCTGTATGGCGATGGAATTCCACAGTCAGCAG 3′ 3′CGCGTGCCAGAGGGCCGCATAGGTAGGGAAGCCG    CATAGGTAGGGAAGCCGGAGCGTTACGACATACCGCTACCTTAAGGTGTCAGTCGTC 5′DNA acceptor strand extended by two cassettes (or bits) (SEQ ID NO 30):     5′  GCGCACGGTCTCCCGGCGTATCCATCCCTTCGGCGTATCCATCCCTTCGGCCTCGCAATGCTGTATGGCGATGGAATTCCACAGTCAGCAG 3′ 3′CGCGTGCCAGAGGGCCGCATAGGTAGGGAA

CATAGGTAGGGAAGCCGGAGCGTTACGACATACCGCTACCTTAAGGTGTCAGTCGTCThis process can be repeated over and over, to extend the DNA strandwith “bits” which encode data.

Experimental Details:

Topoisomerase Charging Reaction: Forty microliters ofstreptavidin-coated magnetic dynabeads (Thermo Fisher) are washed 5times in B & W buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 2 M NaCl). 2.7pmole of biotinylated charging oligonucleotide is added to the beads,and the mixture is incubated for 10 minutes at room temperature, gentlyshaking. The supernatant is drawn off the beads and discarded, leavingonly bound charging oligonucleotide. The beads are then incubated in 1×Cutsmart buffer (New England Biolabs, NEB) containing vaccinia virustopoisomerase (6 ug), 10 units T4 polynucleotide kinase (3′ phosphataseminus, NEB), 0.1 uM ATP (NEB) and 5 mM DTT, for 30 minutes at 37 C tocharge the topoisomerase. After the topoisomerase cleaves the chargingoligonucleotide, the polynucleotide kinase phosphorylates the 5′ end ofthe newly formed by-product oligonucleotide, thereby preventing thetopoisomerase from ligating the charging oligonucleotide back togetheragain, and increasing the efficiency of the charging reaction. Anyuncharged topoisomerase binds to the streptavidin-coated Dynabeads viaelectrostatic forces. The charged topoisomerase is freed from thestreptavidin-coated beads. The polynucleotide kinase (PNK) isneutralized by recombinant shrimp alkaline phosphatase which reversesthe activity of PNK or preferably, the charged topoisomerase is purifiedeither by ion exchange chromatography or via Nickel-NTA beads (thetopoisomerase is His-6 tagged),

Initial binding of Acceptor DNA to beads: Twenty microliters ofstreptavidin-coated magnetic dynabeads (Thermo Fisher) are washed 5times in B&W buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 2 M NaCl). 0.03pmoles of biotinylated DNA acceptor oligonucleotide is added to thewashed beads, and incubated for 10 minutes at room temperature shaking.The supernatant is drawn off of the streptavidin-coated beads anddiscarded, leaving only DNA acceptor strands bound to the beads.

Addition reaction: To prepare addition mix add E coli DNA ligase plus 1mM NAD to the charged topoisomerase. Optional: add 100 uM Coumermycin or1 mM Novobiocin. (rationale: E. coli DNA ligase (which requires NAD)will ‘repair’ the nick left when the charged topoisomerase adds DNA tothe acceptor. This ensures that if an uncharged topoisomerase encountersthis DNA it will not cleave it. Also, coumermycin and novobiocin willinhibit topoisomerase, and preferentially inhibit the ‘charging’ and notthe ‘addition’ reaction. So the inhibitor also helps ensure that anyuncharged topoisomerase formed during the reaction isn't active). Fiftymicroliters of addition mix is added to the beads, and incubated at 37°C. for 15 minutes, to allow for the addition of a DNA cassette to theDNA acceptor molecule. FIG. 76 shows a 4% agarose gel, proving that thecassettes were added as predicted. In order to release the DNA from themagnetic beads to prepare this gel, the sample is digested with Eco(EcoRI)).

The beads are then placed next to a magnet, and the topoisomerasesolution is removed and stored on ice. The beads are then washed 3× withfifty microliters of 1× cutsmart buffer (NEB), to remove any residue ofthe topoisomerase.

Deblocking reaction: The beads are then incubated with fifty microlitersof cutsmart buffer (with NAD and optionally coumermycin/novobiocin),containing 40 units of Bsa I and 1 unit of shrimp alkaline phosphatase.When the restriction enzyme cuts the DNA, a 5′ phosphate is left, andthis inhibits charged topoisomerase from adding another cassette. Theaddition of shrimp alkaline phosphatase removes the 5′ phosphate,thereby effectively de-protecting the cassette. The use of a phosphatasesuch as shrimp alkaline phosphatase or calf intestinal phosphataseenhances the efficiency of the reaction; without the phosphatase, the 5′phosphate has an inhibitory effect.

The Bsa I and SAP are removed from the beads, and the beads are washed3× in cutsmart buffer. They are now ready for the addition of anothercassette by charged topoisomerase.

This sequence of adding a cassette by incubating the DNA acceptor strandwith charged topoisomerase (/ligase), followed by digestion withBsaI/rSAP is repeated for the number of cassettes to be added.

The invention claimed is:
 1. A method for synthesizing a charged polymercomprising at least two distinct monomers or oligomers in ananopore-based device, the nanopore-based device comprising one or moreaddition chambers or channels containing buffer solution and reagentsfor addition of one or more monomers or oligomers to the charged polymerin blocked form, such that only a single monomer or oligomer can beadded in one reaction cycle; and one or more deblocking chambers orchannels containing buffer solution and reagents for removing theblocker group from the charged polymer, wherein the addition chambers orchannels are separated from the deblocking chambers or channels by oneor more membranes comprising one or more nanopores, and wherein thecharged polymer can pass through a nanopore and at least one of thereagents for addition of one or more monomers or oligomers cannot, themethod comprising a) moving the first end of a charged polymer having afirst end and a second end, by electrical attraction, into an additionchamber or channel, whereby monomers or oligomers are added to saidfirst end in blocked form, b) moving the first end of the chargedpolymer with the added monomer or oligomer in blocked form into adeblocker chamber or channel, whereby the blocking group is removed fromthe added monomer or oligomer, and c) repeating steps a) and b), whereinthe monomers or oligomers added in step a) are the same or different,until the desired polymer sequence is obtained.
 2. The method of claim 1wherein the charged polymer is DNA.
 3. The method of claim 1, whereinthe second end of the polymer is bound to a surface.
 4. The method ofclaim 1 wherein the device comprises one or more first addition chambersor channels containing reagents suitable for adding a first type ofmonomer or oligomer and one or more second addition chambers containingreagents suitable for adding a second type of monomer or oligomer, andwherein in step a), the first end of the charged polymer is moved intoeither the first addition chamber or the second addition chamber,depending on whether it is desired to add a first type of monomer oroligomer or a second type of monomer or oligomer.
 5. The method of claim2 wherein the reagents for addition of one or more monomers or oligomersto the charged polymer comprise reagents selected from a topoisomerase,a DNA polymerase, or combinations thereof.